Tannin-containing gastrointestinal formulations and methods of use

ABSTRACT

Tannin-containing compositions and methods of using same to enhance or maintain immune function during simplified nutrition feeding. Pharmaceutical compositions, including enteral nutrition compositions, are provided. The compositions comprise such tannins as proanthocyanidins and/or hydrolysable tannins. Administering the tannins to the gastrointestinal tract of a subject receiving simplified nutrition, such as with enteral nutrition therapy or parenteral nutrition therapy, attenuates or prevents deleterious effects on the gastrointestinal immune system that would otherwise occur with the simplified nutrition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Patent Application 61/751,647 filed Jan. 11, 2013, theentirety of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM053439 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention is directed to tannin-containing compositions fordelivery to the gastrointestinal tract of a subject receiving simplifiednutrition.

BACKGROUND

The gastrointestinal mucosa maintains a physical and chemical barrieragainst 100 trillion resident bacteria as well as food and environmentalantigens. A number of interrelated factors influence this function,including mucus glycoproteins, antimicrobial molecules, specific andnon-specific antibodies, enterocyte tight-junctions, and colonization ofcommensal microbiota. The dietary intake of the host affects the complexinterplay between factors. The route and complexity of nutritionprofoundly influences the mucosal immune system, specifically themucosal associated lymphoid tissue.

Elemental enteral nutrition (EEN) or parenteral nutrition (PN) areuseful therapeutic options for conditions requiring a reduced residualdiet, including pancreatitis, inflammatory bowel disorders such asCrohn's disease, and a variety of other conditions (Ofman et al.Clinical economics review: nutritional support. Ailment Pharmacol Ther1997, 11:453-471; and McClave et al. Journal of Parenteral and EnteralNutrition, 33(3):277-316). A reduction in dietary intake or complexity,such as those that occur with elemental enteral or parenteral nutrition,decreases the number of lymphocytes in Peyer's patches and laminapropria, reduces levels of IgA-stimulating Th-2 type cytokines in thegut wall, and reduces levels of intestinal immunoglobulins (primarilyIgA) compared to chow feeding or administration of a complex enteraldiet containing complex carbohydrates, proteins, and fats. Elementalenteral and parenteral nutrition also increase barrier permeability andsignificantly suppress bacterial diversity within the gut. Elementalenteral or parenteral nutrition thus induce well-defined dysfunction ofthe mucosal immune system, specifically within the gut-associatedlymphoid tissue (GALT), and suppress mucosal barrier function whencompared to normal nutrition. The integrity of the mucosal barrier iscritical for maintaining the physical and chemical barrier againstmicrobes as well as food and environmental antigens. The mucosal barrierdepends on multiple factors including the physical and compositionalcharacteristics of the mucous layer, presence of antimicrobial compoundswithin the mucous layer, immunoglobulin (especially IgA) secretion bythe mucosal immune cells, permeability of the enterocytetight-junctions, and the commensal endogenous microbiota.

Tannins are a class of polyphenolic compounds widely distributed inplant-derived foods and beverages. Tannins are associated withbeneficial health outcomes in epidemiological studies. Tannins arecomposed of two sub-classes, hydrolysable tannins and condensed(proanthocyanidin, PAC) tannins. Tannins have a propensity to bindproteins through hydrogen bonding. For example, proanthocyanidins formcomplexes with salivary glycoproteins, a process that causes astringencyin the oral cavity when many fruits and beverages are ingested. Thiscomplexation increases salivary excretion, hypertrophy of the parotidgland, and a shift in salivary composition to proline-richglycoproteins. Because of poor absorption, a large proportion (sometimesgreater than 95%) of proanthocyanidins remains in the intestinal lumenduring transit. Thus, beneficial dietary effects may occur throughinteractions at the mucosal surface of the gastrointestinal tract, forexample, by influencing secretion of mucins, a class of glycoproteins,in the small intestine.

Mucins are secreted by goblet cells (GC) and play a critical role inmaintaining mucosal integrity. Goblet cells are specialized intestinalepithelial cells. Goblet cells migrate up the villi afterdifferentiating from crypt stem cells and turn over with the epitheliallayer every 3-5 days. Mucin2 (MUC2) is the most abundant mucin secretedby intestinal goblet cells. The importance of MUC2 is underscored inMUC2−/− mice, in which the deficiency leads to the development of lethalcolitis. MUC2 secretion is induced by cholinergic stimulation, while itsproduction is regulated by IL-4 and IL-13 from T-helper 2 cells (Th-2)in the lamina propria or intraepithelial cells.

While the influence of dietary intake or complexity on mucosal barrierand immunity is appreciated, very little is known of the influence of“non-nutritive” dietary compounds, such as tannins. Accordingly,formulations and feeding methods that can counteract the deleteriouseffects of enteral or parenteral nutrition on modulators of mucosalbarrier integrity are needed to provide more efficacious options forpatents that require enteral nutrition, parenteral nutrition, and otherrestricted dietary regimens.

SUMMARY OF THE INVENTION

Compounds that counteract or prevent the deleterious effects of commonlyused enteral or parenteral nutrition formulations have been discoveredand are described herein. Specifically, the administration of varioustypes of tannins, including proanthocyanidins and hydrolysable tannins,has been found to counteract the deleterious effects of enteral orparenteral nutrition on such outcomes as mucosal barrier integrity andmucosal immunity, among others.

Thus, the invention provides an enteral nutrition composition thatincludes a tannin. The composition may include, for example, caloriesfrom simple carbohydrates such as glucose, other monosaccharides, and/ordisaccharides; amino acids; vitamins; minerals; fatty acids and/or fattyacid glyceryl esters; and a tannin. The composition may include, forexample, a protein source comprising at least 20% of the total calories;a carbohydrate source comprising at least 30% of the total calories; anda lipid source comprising at least 30% of total calories; and an amountof tannins. Other possible formulations are described below. The amountof tannins in the composition can be an amount effective to attenuatethe negative effects of enteral nutrition on intestinal barrier functionor mucosal immunity when enteral nutrition is administered to a patient,where such enteral nutrition lacks the tannins described herein.

The enteral nutrition composition can be provided to a patient, forexample, as a tube-fed enteral product. The composition can reduce therisk of diarrhea and other complications that arise from receivingnutrition only from an elemental enteral nutrition product. The enteralnutrition composition that includes tannins as described herein can meetthe nutrient requirements of a variety of patients, such as sepsispatients, trauma, burn or post-surgery patients, and those on aprescribed restricted diet, including intensive care patients, who mayhave compromised absorption capacity, or any medical situation whereenteral nutrition is prescribed, including preparation for surgery.

The invention also provides pharmaceutical compositions comprisingtannins. The pharmaceutical compositions allow for the introduction of abolus of a specially-formulated tannin mixture before/during/afterfeeding of simplified nutrition, such as in enteral or parenteralfeeding. The tannin formulation can be administered orally in solidform. However, tube feeding of liquid forms may be advantageous tocertain patient populations. The methods of administering the tanninscan include concurrent administration (i.e., at the time of simplifiednutrition feeding), prophylactic administration (i.e., introduction oftannins in advance of simplified nutrition feeding), or interventionaladministration (i.e., once problems develop).

Any of the compositions described herein can be substantially free ofmonomeric tannin components, such as single flavan-3-ol units, and/orhydrolysable tannins. The tannins may have degrees of polymerization ofat least 2, at least 3, at least 4 or at least 5. The tannins in thecompositions may be exclusively proanthocyanidins, exclusivelyhydrolysable tannins, or a combination of both. The proanthocyanidinsmay have at least one A-type interflavan bond, or at least one B-typeinterflavan bond.

Accordingly, one version of the invention comprises a composition forenteral nutrition. The composition comprises one or more nutrientcomponents and a tannin. The one or more nutrient components comprise atleast one of a nitrogen source and a carbohydrate source. A lipid sourcemay optionally be included. The nitrogen source is selected from thegroup consisting of individual amino acids and polypeptides. At leastabout 10% by mass of the nitrogen source comprises a nitrogen sourcecomponent selected from the group consisting of individual amino acidsand polypeptides having an average chain length less than about 50residues. The carbohydrate source comprises at least about 10% by massof a carbohydrate source component selected from the group consisting ofmonosaccharides and disaccharides. The one or more nutrient componentsand the tannin may be comprised within a liquid carrier. The tannin maybe present in an amount from about 0.1 mg/L to about 13 g/L.

Another version of the invention comprises a method of ameliorating adeleterious effect on immune function resulting from simplifiednutrition. The method comprises administering an effective amount of atannin to the gastrointestinal tract of a subject receiving a simplifiednutrition composition. The simplified nutrition composition comprisesone or more nutrient components comprising at least one of a nitrogensource and a carbohydrate source. A lipid source may optionally beincluded. The nitrogen source is selected from the group consisting ofindividual amino acids and polypeptides. At least about 10% by mass ofthe nitrogen source comprises a nitrogen source component selected fromthe group consisting of individual amino acids and polypeptides havingan average chain length less than about 50 residues. The carbohydratesource comprises at least about 10% by mass of a carbohydrate sourcecomponent selected from the group consisting of monosaccharides anddisaccharides. The tannin may be administered in an amount of from about1 mg/kg subject body weight per day to about 500 mg/kg subject bodyweight per day. The simplified nutrition composition may be administeredas enteral or parenteral nutrition therapy. The tannin may beadministered in a solid form or in a liquid form. If administered in aliquid form, the tannin may be present in an amount of from about 0.1mg/L to about 13 g/L. The tannin may be administered as formulated inany enteral nutrition composition described herein. The tannin ispreferably administered in an amount effective to increase ileal IL-4,ileal IL-13, goblet cell density, goblet cell size, luminal MUC2concentration, Peyer's patch lymphocytes, STAT6 phosphorylation,polymeric immunoglobulin receptor (pIgR), luminal secretoryimmunoglobulin-A (sIgA), and/or gut microbiota diversity in the subject.

Another version of the invention comprises a method of ameliorating adeleterious effect on immune function resulting from enteral nutritionor parenteral nutrition. The method comprises administering an effectiveamount of a tannin to the gastrointestinal tract of a subject receivingenteral or parenteral nutrition therapy. The tannin may be administeredin an amount of from about 1 mg/kg subject body weight per day to about500 mg/kg subject body weight per day. The tannin may be administered ina solid form or in a liquid form. If administered in a liquid form, thetannin may be present in an amount of from about 0.1 mg/L to about 13g/L. The tannin may be administered as formulated in any enteralnutrition composition described herein. The tannin is preferablyadministered in an amount effective to increase ileal IL-4, ileal IL-13,goblet cell density, goblet cell size, luminal MUC2 concentration,Peyer's patch lymphocytes, STAT6 phosphorylation, polymericimmunoglobulin receptor (pIgR), luminal secretory immunoglobulin-A(sIgA), and/or gut microbiota diversity in the subject.

Various additional non-limiting, exemplary versions of the inventioninclude the following:

Version 1: A composition for enteral nutrition comprising: a tannin andone or more nutrient components comprising at least one of a nitrogensource and a carbohydrate source, wherein: (1) the nitrogen source isselected from the group consisting of individual amino acids andpolypeptides, wherein at least about 10% by mass of the nitrogen sourcecomprises a nitrogen source component selected from the group consistingof individual amino acids and polypeptides having an average chainlength less than about 50 residues; and (2) the carbohydrate sourcecomprises at least about 10% by mass of a carbohydrate source componentselected from the group consisting of monosaccharides and disaccharides.

Version 2: The composition of version 1 wherein the one or more nutrientcomponents comprises the nitrogen source and wherein at least about 10%by mass of the nitrogen source comprises individual amino acids.

Version 3: The composition of version 1 wherein the one or more nutrientcomponents comprises the carbohydrate source and the carbohydrate sourcecomprises at least about 30% by mass of the carbohydrate sourcecomponent selected from the group consisting of monosaccharides anddisaccharides.

Version 4: The composition of version 1 wherein the one or more nutrientcomponents and the tannin are comprised within a liquid carrier.

Version 5: The composition of version 4 wherein the tannin is present inan amount from about 0.1 mg/L to about 13 g/L.

Version 6: The composition of version 1 wherein the tannin comprises aproanthocyanidin.

Version 7: The composition of version 1 wherein the tannin comprises ahydrolysable tannin.

Version 8: The composition of version 1 wherein the composition issubstantially free of monomeric tannin components.

Version 9: A method of ameliorating a deleterious effect on immunefunction resulting from simplified nutrition comprising administering aneffective amount of a tannin to the gastrointestinal tract of a subjectreceiving a simplified nutrition composition, wherein the simplifiednutrition composition comprises one or more nutrient componentscomprising at least one of a nitrogen source and a carbohydrate source,wherein: (1) the nitrogen source is selected from the group consistingof individual amino acids and polypeptides, wherein at least about 10%by mass of the nitrogen source comprises a nitrogen source componentselected from the group consisting of individual amino acids andpolypeptides having an average chain length less than about 50 residues;and (2) the carbohydrate source comprises at least about 10% by mass ofa carbohydrate source component selected from the group consisting ofmonosaccharides and disaccharides.

Version 10: The method of version 9 wherein the simplified nutritioncomposition is administered to the subject via a tube directly to thegastrointestinal tract.

Version 11: The method of version 9 wherein the simplified nutritioncomposition is administered to the subject parenterally.

Version 12: The method of version 9 wherein the tannin comprises aproanthocyanidin.

Version 13: The composition of version 9 wherein the tannin comprises ahydrolysable tannin.

Version 14: The composition of version 9 wherein the composition issubstantially free of monomeric tannin components.

Version 15: The method of version 9 comprising administering the tanninin an amount of from about 1 mg/kg subject body weight per day to about500 mg/kg subject body weight per day.

Version 16: The method of version 9 wherein the tannin is administeredin a solid form.

Version 17: The method of version 9 wherein the tannin is administeredin a liquid form.

Version 18: The method of version 17 wherein the tannin is present in anamount of from about 0.1 mg/L to about 13 g/L.

Version 19: The method of version 9 wherein the simplified nutritioncomposition, when administered in the absence of the tannin, decreasesat least one of ileal IL-4, ileal IL-13, goblet cell density, gobletcell size, luminal MUC2 concentration, Peyer's patch lymphocytes, STAT6phosphorylation, polymeric immunoglobulin receptor (pIgR), luminalsecretory immunoglobulin-A (sIgA), and gut microbiota diversity in thesubject.

Version 20: The method of version 9 wherein the effective amount of thetannin is an amount effective to increase at least one of ileal IL-4,ileal IL-13, goblet cell density, goblet cell size, luminal MUC2concentration, Peyer's patch lymphocytes, STAT6 phosphorylation,polymeric immunoglobulin receptor (pIgR), luminal secretoryimmunoglobulin-A (sIgA), and gut microbiota diversity in the subject.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of chow (n=11), elementary enteral nutrition(EEN) (n=14), EEN+low proanthocyanidin (lowPAC; 8 mg gallic acidequivalents of PAC/kg body weight) (n=14), EEN+mid proanthocyanidin(midPAC; 50 mg gallic acid equivalents of PAC/kg body weight) (n=14),and EEN+high proanthocyanidin (highPAC; 100 mg gallic acid equivalentsof PAC/kg body weight) (n=14) feeding on ileal tissue IL-4 levels.

FIG. 2 depicts the effect of chow (n=6), EEN (n=8), EEN+lowPAC (n=8),EEN+midPAC (n=8), and EEN+highPAC (n=5) feeding on ileal tissue IL-13levels.

FIG. 3 depicts the effect of chow (n=8), EEN (n=12), EEN+lowPAC (n=8),EEN+midPAC (n=7), and EEN+highPAC (n=10) feeding on ileal goblet celldensity.

FIG. 4 depicts the effect of chow (n=6), EEN (n=7), EEN+lowPAC (n=6),EEN+midPAC (n=8), and EEN+highPAC (n=7) feeding on ileal goblet cellsize.

FIG. 5 depicts the effect of chow (n=9), EEN (n=10), EEN+lowPAC (n=7),EEN+midPAC (n=9), and EEN+highPAC (n=8) feeding on luminal MUC2.

For FIGS. 1-5, the data is presented as mean±SEM, and the superscript“a” denotes significance of difference when compared to chow feeding,while the superscript “b” denotes significance of difference whencompared to EEN feeding alone.

FIG. 6 depicts the total number of Peyer's Patch lymphocytes in chow,EEN, and EEN+proanthocyanidin (PAC) fed mice. *P<0.001 vs. EEN.

FIG. 7 depicts ileum tissue IL-4 levels in chow, EEN, and EEN+PAC fedmice. *P<0.05 vs. EEN.

FIG. 8A depicts ileum tissue phosphorylated STAT6 (Tyr 641) levels inchow, EEN, and EEN+PAC fed mice. FIG. 8B depicts ileum tissuephosphorylated STAT6 (Tyr 645) levels in chow, EEN, and EEN+PAC fedmice. *P<0.01 vs. EEN. # P<0.05 vs. EEN.

FIG. 9 depicts ileum tissue levels of polymeric immunoglobulin receptor(pIgR) in Chow, EEN, and EEN+PAC fed mice. *P<0.05 vs. EEN. # P<0.05 vs.EEN.

FIG. 10 depicts concentration of secretory IgA in small intestineluminal wash samples in Chow, EEN, and EEN+PAC fed mice. *P<0.01 vs.EEN. # P<0.05 vs. EEN.

FIGS. 11A-C depict peaks observed greater than 0.5% of total signal asdetermined from automated ribosomal intergenic spacer analysis (ARISA)in intestinal content from the ileal-cecal junction of chow (FIG. 11A),EEN (FIG. 11B), and EEN+PAC (FIG. 11C) fed mice.

FIG. 12 depicts a dendrogram illustrating microbiome Jaccard'ssimilarity coefficients between chow, EEN, and EEN+PAC fed mice. Theresults illustrate the greatest similarity between chow and EEN+PAC.

FIG. 13 depicts the results of principal coordinate analysis of chow,EEN, and EEN+PAC fed mice. Ellipses are 95% confidence.

DETAILED DESCRIPTION OF THE INVENTION

The gastrointestinal mucosa maintains a physical and chemical barrieragainst 100 trillion resident bacteria as well as food and environmentalantigens. The dietary intake of a host affects the complex interplayamong the many factors that influence the function of this barrier. Areduction in dietary intake or complexity, such as that which occurswith parenteral nutrition (i.e., IV feeding) or enteral nutrition (i.e.,tube feeding), reduces the cellular and molecular functionality of themucosal immune system in the intestines. Accordingly, enteral orparenteral nutrition significantly suppresses the mucosal immune systemand increases the risk of infections and related complications, byincreasing mucosal barrier permeability, and suppressing bacterialdiversity within the gut.

Tannins are complex oligomeric polyphenolic compounds widely distributedin fruits, including grapes, cranberries, and apples, and other foodsand beverages such as chocolate and wine. As described in the examplesthat follow, tannin preparations were isolated from various sources andadded to an elemental enteral nutrition formulation. Adding the tanninto the elemental enteral nutrition formulation was found to counteractthe deleterious effect of elemental enteral nutrition on modulators ofmucosal barrier integrity.

The chemical nature of the tannins used in the compositions describedherein allow the tannins to associate with the mucous layer of thegastrointestinal tract, delaying their clearance and thus providing along-lasting beneficial effect on the immune system between enteral orparenteral feedings. Furthermore, due to the complexity and diversity oftannin chemistry, defined tannin preparations with defined biologicaleffects can be prepared for use in enteral nutrition formulations. Asthe gastrointestinal tract poorly absorbs these plant-derived tannins,they are not believed to have problems associated with systemictoxicity.

Some current enteral compositions, such as the enteral product describedby U.S. Pat. No. 5,229,136 (Mark et al.), provide an enteral product forreducing diarrhea in tube-fed patients. The product includes specificnutrient blends, including a high fiber content (greater than 14 g/mL),wherein the fiber can include components such as pectin, carob pods, andtannin-enriched extracts of carob pod. The high fiber content of theproduct (because of the amount of fiber/mL) would provide a highlyviscous composition that may be problematic for the general tube-fedpatient population. The tannin compositions described herein canalleviate these problems.

DEFINITIONS

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14th Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moretannins can refer to one or two, one to three, one to five, or one toabout ten, different tannins, for example, having different degrees ofpolymerization.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percents, proximate to the recited range that are equivalent interms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

The term “patient” or “subject” refers to any animal, such as a mammal,including mice, rats, other rodents, rabbits, dogs, cats, swine, cattle,sheep, horses, and/or primates, for example, humans.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an effective amount can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a compound described herein, or anamount of a combination of compounds described herein, e.g., that iseffective to treat or prevent a disease or disorder, or to treat thesymptoms of the disease or disorder, in a subject. Thus, an “effectiveamount” generally means an amount that provides the desired effect.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic condition or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, or condition. The inhibition can be greater than about 20%,40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth orprogression that occurs in the absence of the treatment or contacting.

In reference to tannins, the phrase “substantially free of monomericcomponents” means that the tannins are at least dimeric in compositionand few or no monomeric tannin species are present in the sample. Forexample, a tannin that is substantially free of monomeric components caninclude less than about 5 wt. % monomeric tannins, less than about 3 wt.% monomeric tannins, less than about 1 wt. % monomeric tannins, lessthan about 0.5 wt. % monomeric tannins, less than about 0.1 wt. %monomeric tannins, or no monomeric tannins. An example of a monomerictannin is the compound tannic acid (pentagalloyl-D-glucose (C₇₆H₅₂O₄₆,mw=1701.18)). Other compounds that can be specifically included orexcluded from the composites described herein include catechin(mw=290.26), quercetin (mw=302.24), cyanidin (mw=287.24), gelatin,epichlorohydrin moieties, or combinations thereof.

Improving mucosal immune function and mucosal barrier function in apatient refers to reducing factors that produce a changes associatedwith the suppression of mucosal immune function, including lower levelsof Th2 stimulating cytokines, IL 4 and IL 13, lower goblet cell densityand size, lower luminal MUC2 levels in the ileum, lower numbers ofPeyer's patch lymphocytes, lower levels of STAT6 phosphorylation, lowerlevels of polymeric immunoglobulin receptor (pIgR), lower levels ofluminal secretory immunoglobulin-A (sIgA), and/or reduced gut microbiotadiversity. Thus, improving mucosal immune function and mucosal barrierfunction in a patient can refer to increasing levels of the Th2stimulating cytokines, IL-4 and IL-13, increasing goblet cell densityand size, increasing luminal MUC2 levels in the ileum, increasingnumbers of Peyer's patch lymphocytes, increasing levels of STAT6phosphorylation, increasing levels of polymeric immunoglobulin receptor(pIgR), increasing levels of luminal secretory immunoglobulin-A (sIgA),and/or increasing gut microbiota diversity.

Mucosal system functional indicators can include fecal orgastrointestinal luminal levels and/or composition of mucins, secretoryimmunoglobulin A (sIgA) or microbiome.

Enteral nutrition refers to nutritional support given via the alimentarycanal or any route connected to the gastrointestinal system (i.e., theenteral route), for example, where nutrients are administered in aliquid carrier directly into the gastrointestinal (GI) tract, mostcommonly through a tube (i.e., tube feeding). Enteral nutrition (EN) isoften used to prevent progressive malnutrition in patients withcontraindication to normal feeding. Elemental enteral nutrition (EEN) isa nutritional support strategy utilized to treat patients withgastrointestinal disorders and inflammatory bowel disease, for example,typically with a drastically simplified diet that eliminates thecomplexity offered by whole foods and beverages. Elemental enteralnutrition suppresses the mucosal immune system by decreasing Peyer'spatch density, lamina propria lymphocytes, Th2 cytokines, and IgA whileincreasing mucosal barrier permeability. The mucosal barrier isregulated in-part by mucins secreted by specialized epithelial cellscalled goblet cells (GC). The primary goblet cell mucin is mucin2 (MUC2)and deficiency of MUC2 leads to lethal colitis. MUC2 production isstimulated by the Th2 cytokines IL-4 and IL-13.

Parenteral nutrition (PN) refers to compositions and the administrationthereof where nutrients are provided parenterally (e.g., intravenously)such that the nutrients bypass the gastrointestinal tract completely.Unfortunately parenteral nutrition feeding is associated with anincreased risk of infection compared to enteral diets, especially in thecritically ill, thus improvements in the current formulations areneeded.

Tannins

Tannins include oligomeric polyphenols that occur naturally in a varietyof plants. Isolated tannins typically form a heterogeneous mixture oftannin compounds. Tannin compounds can be subdivided into two groups:condensed tannins, also known as proanthocyanidins (PAC), andhydrolysable tannins (HT). The tannins used in the compositionsdescribed herein may comprise any one or more proanthocyanidin, any oneor more hydrolysable tannin, or any combination of one or moreproanthocyanidin and one or more hydrolysable tannin.

Proanthocyanidins are complex oligomeric polyphenolics widelydistributed in plant sources such as cranberries and other sources.Tannin oligomers typically occur as dimers, trimers, tetramers,pentamers, hexamers, heptamers, octamers, nonamers, or decamers.Oligomers with greater than ten monomeric segments can also be isolatedor synthesized, such as oligomers that include up to 50 units, asdescribed herein. For a review of tannin nomenclature, see Beecher (J.Nutrition 2003, 3248S-3254S), which is incorporated herein by reference.In some embodiments, certain monomeric tannins or tannins with a lowdegree of polymerization can be excluded from a particular composition.For example, a composition may exclude catechin, tannic acid, or othermonomeric tannins, dimeric tannins, trimers, or tetramers, PA tannins,or alternatively, hydrolysable tannins, a certain molecular weight rangeof tannins, or a type, class, or specific tannin cited in Beecher.

Proanthocyanidins are polymers of flavan-3-ols and flavans linkedthrough interflavan bonds. Proanthocyanidins can have various types ofinterflavan linkages, including B-type and A-type linkages. B-typeinterflavan linkages are defined by the presence of C4→C8 or C4→C6interflavan bonds. A-type interflavan linkages are defined by thepresence of C4→C8 and C2→O→C7 interflavan bonds. The linkages can be αor β.

Monomers that may polymerized in the proanthocyanidins include, withoutlimitation, catechin, epicatechin, epigallocatechin, epicatechingallate, epigallocatechin gallate, epiafzelechin, fisetinidol,guibourtinidol, mesquitol, and robinetinidol, among others. Variousflavonols may also be included as monomers in the proanthocyanidins,particularly as terminal units.

Various proanthocyanidin subtypes include, without limitation,prodelphinidins, proguibourtinidins, prorobinetinidins,proteracacinidins, and profisetinidins, among others.

The proanthocyanidins may be glycosylated with any glycone moiety at oneor more positions, such as on an otherwise pendant hydroxyl group. Typesof glycone moieties include, without limitation, glycopyranosylglycones, furanosyl glycones, oligosaccharides (diglycosides,triglycosides, etc.), and amino glycone derivatives. Examples ofglycopyranosyl structures include glucuronic acid, glucose, mannose,galactose, gulose, allose, altrose, idose, and talose. Examples offuranosyl structures include those derived from fructose, arabinose, orxylose. Examples of diglycosides (i.e., glycone moieties with 2 glyconeunits) include sucrose, cellobiose, maltose, lactose, trehalose,gentiobiose, and melibiose. Examples of triglycosides (i.e., glyconemoieties with 3 glycone units) include raffinose or gentianose. Examplesof amino derivatives include N-acetyl-D-galactosamine,N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, N-acetylneuraminic acid,D-glucosamine, lyxosylamine, D-galactosamine, and the like.

Other chemical modifications of the pendant hydroxyl groups areacceptable.

The proanthocyanidins can have any degree of polymerization, such as 1to about 50, 1 to about 25, 1 to about 20, 1 to about 12, 1 to about 10,2 to about 50, 2 to about 25, 2 to about 20, 2 to about 12, 2 to about10, or any range between any integers from 1 to about 50, including anyrange between any integers from 2 to about 50.

At least a subset of the proanthocyanidins suitable for use in thepresent invention may comprise one or more units of:

wherein:

------ is a single or double bond;

R⁵ and R^(3′)-R^(5′) are each independently —H, —OH, —O-glycoside,alkyloxy, alkanoyl, or alkanoyloxy;

R⁴ is —H, ═O, a C4-C6 inter-unit bond, or a C4-C8 inter-unit bond;

R⁶ is —H or C6-C4 inter-unit bond;

R⁷ is —H, —OH, —O-glycoside, alkyloxy, alkanoyl, or alkanoyloxy, or aC7-O—C2 inter-unit bond;

R⁸ is —H, —OH, —O-glycoside, alkyloxy, alkanoyl, or alkanoyloxy, or aC8-C4 inter-unit bond

R² is H or a C2-O—C7 inter-unit bond;

R³ is —H, —OH, —O-glycoside, alkyloxy, alkanoyl, alkanoyloxy, or:

-   -   wherein R⁹-R¹³ are each independently —H, —OH, —O-glycoside, or        alkyloxy, alkanoyl, or alkanoyloxy, provided that R⁹-R¹³ are not        simultaneously hydrogen.

Scheme 1 illustrates an exemplary cranberry polyflavan-3-ol showingstructural variation in the nature of interflavan linkage andsubstitution to an anthocyanin terminal unit through a CH₃—CH bridge.

Scheme 1 shows a representative structure of a proanthocyanidin dimerlinked to an anthocyanin through an ethyl (methine methyl) group.Variation in degree of polymerization, position and number of A-typeversus B-type interflavan bonds and substitutions with anthocyaninsleads to large structural heterogeneity among PAC oligomers.

Scheme 2 illustrates two sub-types of proanthocyanidin: procyanidins andprodelphinidins (for the trimer x=1; for the tetramer, x=2; for thepentamer, x=3; for the hexamer, x=4; for the heptamer, x=5; for theoctamer, x=6; for the nonamer, x=7; and for the decamer, x=8).Procyanidins (R=H) contain catechin and/or epicatechin (CE) subunits;prodelphinidins (R=OH) contain gallocatechin and/or epigallocatchin (GE)subunits.

In various proanthocyanidins, the R groups of Scheme 2 can eachindependently be H or OH. In some embodiments, one or more hydroxylgroups may be glycosylated. In some embodiments, x is 1 to about 50, 1to about 25, 1 to about 20, 1 to about 12, 1 to about 10, or a range ofbetween any integers from 1 to 50.

Other proanthocyanidin tannins include glycosylated heteropolyflavans,such as those illustrated in Scheme 3. Representative compounds shown inScheme 3 include proluteolinidin (R1=OH); proapigininidin (R1=H);eriodictyol (R2=H); and eriodictyol 5-O-β glucoside (R2=glucose).Krueger et al. have described a variety of known heteropolyflavans-3-olsand glycosylated heteropolyflavans (see J. Agric. Food Chem. 2003, 51,538-543, which is incorporated herein by reference).

where R1 is H or OH; R2 is H or glucose; and glu is glucose (e.g., aβ-glucoside).

In some embodiments, x of Scheme 3 is 1 to about 50, 1 to about 25, 1 toabout 20, 1 to about 12, 1 to about 10, or a range of between any tointegers from 1 to 50. Several examples of condensed tannins aredescribed in U.S. Pat. No. 7,122,574 (Romanczyk et al.), which isincorporated herein by reference. A review by Reed et al. (Phytochem.66(18): 2248-2263 (2005)) describes the structural heterogeneity oftannin polyphenols from cranberries, grape seed extracts, sorghum, andpomegranates as characterized by MALDI-TOF MS. Examples of plants thatproduce proanthocyanidins include cranberries, blueberries, grapes,sorghum, and pine.

Scheme 4 shows an additional example of a proanthocyanidin, which is anoligomer of three flavan-3-ol subunits.

A review by Reed et al. (Phytochem. 66(18): 2248-2263 (2005)) describesthe structural heterogeneity of tannin polyphenols from cranberries,grape seed extracts, sorghum, and pomegranates as characterized byMALDI-TOF MS. Examples of plants that produce proanthocyanidins includecranberries, blueberries, grapes, sorghum, and pine.

The proanthocyanidins suitable for use in the invention may comprise anyproanthocyanidin described herein, known in the art, found in nature, orsynthesized in a laboratory. A method for synthesizing proanthocyanidinsis described in U.S. Pat. No. 6,420,572 to Romanczyk Jr. et al.

Hydrolysable tannins include esters of polyol core moieties, such assugars. The sugar is usually D-glucose but may include other sugars suchas cyclitols, quinic acid, shikimic acids, glucitol, hammamelose, andquercitol, among others. The hydroxyl groups of the sugar are partiallyor totally esterified with phenolic groups such as gallic acid,polymeric galloyl esters thereof, and/or oxidatively cross-linkedgalloyl groups, such as ellagic acid and gallagic acid. Gallotannins,for example, are polygalloyl esters, and ellagitannins are ellagic acidesters. Hydrolyzable tannins can be hydrolyzed by weak acids or weakbases to produce carbohydrate and phenolic acids.

Scheme 5 illustrates a pomegranate ellagitannin showing structuralvariation in nature of esterification of the glucose core molecule.

Hydrolysable tannins, such as the compound shown in Scheme 5, can beisolated in oligomeric forms that include 2 to about 12 hydrolysabletannin moieties, for example, linked by oxidative C—O coupling betweengalloyl and hexahydroxydiphenoyl moieties of the monomeric precursors.Common coupling also occurs between two ellagic acid moieties, or byaddition of gallic acid moieties to the saccharide core of an oligomer.See Quideau and Feldman, Chem. Rev. 1996, 96, 475-503, which isincorporated herein in its entirety.

Accordingly, in some embodiments of compositions described herein, thehydrolysable tannins employed will be oligomeric hydrolysable tannins.Thus, in some embodiments, oligomeric hydrolysable tannins include atleast two saccharide core moieties. Other embodiments can includemonomeric hydrolysable tannins, and yet other embodiments can excludemonomeric tannins. In some embodiments, a hydrolysable tannin willinclude one or more (e.g., 1, 2, 3, 4, 5, or more) ellagic acidmoieties, and in some embodiments, a hydrolysable tannin will includeone or more (e.g., 1, 2, 3, 4, 5, or more) gallagic acid moieties.

Examples of plants that produce hydrolysable tannins includepomegranates, strawberries, raspberries, blackberries, sumac (Rhuscoriaria), chestnut wood (Castanea sativa), oak wood (Quercus robur,Quercus petraea and Quercus alba), tara pods (Caesalpinia spinosa),gallnuts (Quercus infectoria and Rhus semialata), myrobalan (Terminaliachebula), and Aleppo gallnuts (Andricus kollari), among others.Significant quantities of hydrolysable tannins can be isolated from, forexample, pomegranate husks. Specific hydrolysable tannins frompomegranates include punicalin and punicalagin (the alpha or beta isomerof 2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose, with amolecular weight of 1084) and stereochemical isomers thereof (see Martinet al. J. Sci. Food. Agric. 2009, 89:157-162). Other hydrolysabletannins are described by Quideau and Feldman (Chem. Rev. 1996, 96,475-503).

As with the proanthocyanidins, the pendent hydroxyl groups on thecondensed tannins may be glycosylated or otherwise modified.

The tannins suitable for use in the invention may comprise anyproanthocyanidin or hydrolysable tannin described herein, known in theart, found in nature, or synthesized or modified in a laboratory.Methods for synthesizing proanthocyanidins and hyrolysable tannins areknown in the art. See, e.g., U.S. Pat. No. 6,420,572 to Romanczyk Jr. etal.

Enteral Tannin Compositions and Administration Thereof

The tannins described herein can be added in effective amounts toenteral formulations and administered to a patient during enteralnutrition therapy using standard techniques, such as through thenasogastric system.

The enteral compositions may comprise one or more of a nitrogen source,a carbohydrate source, and a lipid source.

The nitrogen source is preferably in the form of individual amino acidsand/or polypeptides. Exemplary nitrogen sources include casein,caseinates, soy protein, whey protein, lactalbumin, milk proteinconcentrate, hydrolyzed casein, hydrolyzed soy protein, hydrolyzed wheyprotein, hydrolyzed lactablumin, hydrolyzed milk protein concentrateand/or crystalline L-amino acids.

In elemental enteral formulations, the nitrogen source is exclusivelycomprised of individual amino acids or primarily comprised of individualamino acids and polypeptides having a short chain length. In variousversions of the invention, for example, at least about 1% by mass, atleast about 5% by mass, at least about 10% by mass, at least about 20%by mass, at least about 30% by mass, at least about 40% by mass, atleast about 50% by mass, at least about 60% by mass, at least about 70%by mass, at least about 80% by mass, at least about 90% by mass, atleast about 95% by mass, or at least about 99% by mass of the nitrogensource is comprised of either individual amino acids or individual aminoacids in combination with polypeptides having, on average, a short chainlength. As used herein, “short chain length” may refer to a chain lengthless than about 50 residues, less than about 40 residues, less thanabout 30 residues, less than about 20 residues, less than about 10residues, less than about 5 residues, or less than about 3 residues.Methods of determining the proportion and size of amino acids and/orpolypeptides in a composition are known in the art.

If present in the composition, the nitrogen source may comprise up toabout 99% of the caloric content of the composition, such as about 1-99%of the caloric content of the composition, about 10-70% of the caloriccontent of the composition, or about 15-60% of the caloric content ofthe composition. Some compositions of the invention may be substantiallydevoid of a nitrogen source, such as amino acids or polypeptides.

The carbohydrate source may comprise any combination of simplecarbohydrates (i.e., monosaccharides and disaccharides) and complexcarbohydrates. Exemplary carbohydrate sources include glucose, sucrose,fructose, dextrose, glucose polymers, starches such as corn starch orhydrolyzed cornstarch, dextrin, maltodextrin, and sugar alcohols.

Compositions for elemental enteral nutrition preferably have a highproportion of simple carbohydrates. In various versions of theinvention, for example, at least about 1% by mass, at least about 5% bymass, at least about 10% by mass, at least about 20% by mass, at leastabout 30% by mass, at least about 40% by mass, at least about 50% bymass, at least about 60% by mass, at least about 70% by mass, at leastabout 80% by mass, at least about 90% by mass, at least about 95% bymass, or at least about 99% by mass of the carbohydrate source iscomprised of monosaccharides or a combination of monosaccharides anddisaccharides.

If present in the composition, the carbohydrate source may comprise upto about 99% of the caloric content of the composition, such as about1-99% of the caloric content of the composition, about 10-80% of thecaloric content of the composition, or about 20-70% of the caloriccontent of the composition. Some compositions of the invention may besubstantially devoid of a carbohydrate source.

The lipid source preferably comprises free fatty acids and/or glycerides(i.e., monoglycerides, diglycerides, and triglycerides). Exemplary lipidsources include fatty acid esters, fish oil, medium chain triglycerides,safflower oil, sardine oil, soybean oil, soy lecithin, structuredlipids, borage oil, canola oil, corn oil, fish oil, high oleic sunfloweroil, medium chain triglycerides, menhaden oil, mono- and diglycerides,palm kernel oil, safflower oil, soybean oil, soy lecithin, omega-3 fattyacids, and omega-6 fatty acids.

Compositions for elemental enteral nutrition are preferably low in fatwith a small proportion of calories being derived from long-chain fattyacid lipids. In various versions of the invention, for example, thelipid source comprises less than about 1% by mass, less than about 5% bymass, less than about 10% by mass, less than about 20% by mass, lessthan about 30% by mass, less than about 40% by mass, less than about 50%by mass less than about 60% by mass, less than about 70% by mass, lessthan about 80% by mass, less than about 90% by mass, less than about 95%by mass, or less than about 99% by mass of long-chain fatty acid lipids.In some versions, the lipid source is substantially devoid of long-chainfatty acid lipids.

Compositions for elemental enteral nutrition typically replacelong-chain fatty acid lipids with short- or medium-chain fatty acidlipids. Accordingly, in various versions of the invention, at leastabout 1% by mass, at least about 5% by mass, at least about 10% by mass,at least about 20% by mass, at least about 30% by mass, at least about40% by mass, at least about 50% by mass, at least about 60% by mass, atleast about 70% by mass, at least about 80% by mass, at least about 90%by mass, at least about 95% by mass, or at least about 99% by mass ofthe lipid source is comprised of short- or medium-chain fatty acidlipids.

As used herein, “short-chain fatty acid lipid” refers to a fatty acidmoiety-containing lipid (i.e., free fatty acid, cholesterol ester,phospholipid, glyceride, etc.) with all fatty acid moieties containingfewer than 6 aliphatic carbons; “medium-chain fatty acid lipid” refersto a fatty acid moiety-containing lipid with all fatty acid moietiescontaining fewer than 12 aliphatic carbons; and “long-chain fatty acidlipid” refers to a fatty acid moiety-containing lipid with at least onefatty acid moiety comprising more than 12 aliphatic carbons.

If present in the composition, the lipid source may comprise up to about99% of the caloric content of the composition, such as about 1-99% ofthe caloric content of the composition, about 10-70% of the caloriccontent of the composition, or about 20-60% of the caloric content ofthe composition. Some compositions of the invention may be substantiallydevoid of a lipid source.

The various proportions of the nitrogen source, the carbohydrate source,and the lipid source may be adjusted based on the nutritional or medicalneeds of the subject. For example, some compositions may comprise about40% of caloric content as a carbohydrate source, about 20% of caloriccontent as a nitrogen source, and about 40% of caloric content as alipid source. Others may comprise about 60% of caloric content as acarbohydrate source, about 30% of caloric content as a nitrogen source,and about 20% of caloric content as a lipid source. Others may compriseabout 80% of caloric content as a carbohydrate source, about 10% ofcaloric content as a nitrogen source, and about 10% of caloric contentas a lipid source. Certain enteral compositions can contain about 17-25%of the total calories from a nitrogen source, about 35-50% of totalcalories from a nitrogen source, and about 25-48% of total calories froma carbohydrate source.

The caloric density of the enteral nutrition composition is preferablyat least about 1 Kcal/mL, at least about 1.2 Kcal/mL, at least about 1.3Kcal/mL, at least about 1.5 Kcal/mL, or about 1.3-1.5 Kcal/mL.

The enteral nutrition composition may comprise proanthocyanidins,hydrolysable tannins, or a mixture of both.

The enteral nutrition composition of the invention can be provided inliquid form or dehydrated (powder) form, the latter of which may bemixed with a liquid carrier, such as water, before administering.

The enteral nutrition composition may comprise any effective amount ofthe tannin. Exemplary amounts of tannin in a liquid form of thecomposition include at least about 0.01 mg/L, at least about 0.1 mg/L,at least about 0.5 mg/L, at least about 1 mg/L, at least about 5 mg/L,or at least about 10 mg/L. Exemplary amounts of tannin in a liquid formof the composition may additionally or alternatively include less thanabout 15 g/L, less than about 13 g/L, less than about 10 g/L, less thanabout 1 g/L, or less than about 100 mg/L. If the composition is providedin dehydrated form, the composition preferably comprises an amount oftannin to comprise the above-listed concentrations when mixed with anappropriate amount solvent or liquid carrier for the desired caloricneeds of the subject. Exemplary amounts of tannin in a dehydrated forminclude at least about 0.01% w/v, at least about 0.1% w/v, at leastabout 0.5% w/v, at least about 1% w/v, at least about 5% w/v, or atleast about 10% w/v. Exemplary amounts of tannin in a dehydrated form ofthe composition may additionally or alternatively include less thanabout 100 g/L, less than about 50% w/v, less than about 25% w/v, lessthan about 15% w/v, less than about 13% w/v, less than about 10% w/v,less than about 1% w/v, or less than about 0.1% w/v.

In addition to the tannins, the enteral nutrition composition caninclude fiber, such as insoluble soy polysaccharide, insoluble pectin,hydrolyzed plant gums, carob pod, and/or extracts of carob pods. Thecomposition can also optionally include one or more of the vitamins andminerals recommended by the US RDA. Exemplary vitamins and minerals areprovided in the following examples. The composition can also includeadditional additives that can aid the health or immune function of asubject and benefit the subject's digestive tract. Such additives caninclude, for example, cellulose fiber, IL-25, rutin, D(+)-catechin,ellagic acid, quercetin, or curcumin, for elevating immunoglobulin A.

Examples of products that can be combined with the tannins describedherein include the enteral formulations described by U.S. Pat. No.5,229,136 to Mark et al.; U.S. Pat. No. 5,723,446 to Gray et al.; U.S.Pat. No. 7,196,065 to Ernest; U.S. Pat. No. 7,790,209 to Ohmori et al.;U.S. Pat. No. 7,758,893 to Hageman et al.; Makola, Elemental andSemi-Elemental Formulas: Are They Superior to Polymeric Formulas?Practical Gastroenterology, December 2005, in Nutrition Issues inGastroenterology, Series #34, Ed. Parrish; and Malone, Enteral FormulaSelection: A Review of Selected Product Categories. PracticalGastroenterology, June 2005, in Nutrition Issues in Gastroenterology,Series #28, Ed. Parrish.

The enteral nutrition composition may be administered via a tube intothe gastrointestinal tract that extends at least through the mouth orthat extends at least through the mouth and throat.

Pharmaceutical Compositions and Administration Thereof

The tannins described herein can be formulated as pharmaceuticalcompositions and administered to a mammalian subject, such as a humanpatient, in a variety of forms. The forms can be adapted foradministration either orally or rectally to the gastrointestinal tract.The tannins can be administered in either a solid or a liquid form.

The pharmaceutical compositions may be prepared by any of the methodswell known in the art of pharmacy. All methods include the step ofbringing the tannins into association with a pharmaceutically acceptablecarrier. A carrier is pharmaceutically acceptable if compatible withother ingredients in the particular composition and not deleterious tothe recipient thereof. In general, the compositions are prepared byuniformly and intimately bringing the tannins into association with aliquid or solid carrier and then, if necessary, shaping the product intoa desired unit dosage form.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented in a discrete solid form, e.g., ascapsules, hard or soft shell gelatin capsules, troches, cachets,tablets, boluses, wafers, lozenges and the like, each containing apredetermined amount of the tannin; in powder or granular form; or inliquid form, e.g., as a collyrium, suspension, solution, syrup, elixir,emulsion, dispersion and the like. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing in a suitable machinethe tannins in a free-flowing form, e.g., a powder or granules,optionally mixed with accessory ingredients or excipients, e.g.,binders, lubricants, inert diluents, surface active or dispersingagents. Molded tablets may be made by molding in a suitable machine, amixture of the powdered tannins with any suitable carrier.Pharmaceutical compositions for oral administration may comprise any ofthe enteral nutrition compositions described above.

Compositions suitable for rectal administration may comprise asuppository, preferably bullet-shaped, containing the tannins and apharmaceutically-acceptable carrier therefor such as hard fat,hydrogenated cocoglyceride, polyethylene glycol and the like.Compositions suitable for rectal administration may alternativelycomprise the tannin and pharmaceutically-acceptable liquid carrierstherefor such as 50% aqueous ethanol or an aqueous salt solution whichis physiologically compatible with the rectum or colon.

In addition to the aforementioned ingredients, the compositions of thisinvention may further include one or more optional accessoryingredients(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, colorants, binders, surfactants,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like. For example, the tablets, troches, pills,capsules, and the like may also contain one or more of the following:binders such as gum tragacanth, acacia, corn starch or gelatin;excipients such as dicalcium phosphate; a disintegrating agent such ascorn starch, potato starch, alginic acid and the like; and a lubricantsuch as magnesium stearate. A sweetening agent such as sucrose,fructose, lactose or aspartame; or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring, may be added. When the unitdosage form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac or sugar and the like. A syrup or elixir may containsucrose or fructose as a sweetening agent, methyl and propyl parabens aspreservatives, a dye, and flavoring such as cherry or orange flavor. Anymaterial used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the tannin may be incorporated intosustained-release preparations and devices.

The pharmaceutical compositions may comprise the tannin in unit dosageform. The term “unit dosage” or “unit dose” is denoted to mean apredetermined amount of the tannin sufficient to be effective fortreating each of the indicated activities. Preferred unit dosageformulations are those containing a daily dose, daily sub-dose, or anappropriate fraction thereof, of the tannin. The percentage of thetannin in the pharmaceutical compositions can vary and may convenientlybe from about 0.1% to about 90%, about 2% to about 60%, or about 5% toabout 20%, of the weight of a given unit dosage form.

Useful dosages of the tannins described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a tannin required foruse in treatment will vary not only with the particular degree ofpolymerization selected but also with the route of administration, thenature of the condition being treated, and the age and condition of thepatient, and will be ultimately at the discretion of an attendantphysician or clinician.

The tannin may be administered to the subject in an amount of from about1 μg/kg body weight per day to about 500 mg/kg body weight per day, suchas about 0.1 to about 200 mg/kg body weight per day or about 1 to about100 mg/kg body weight per day. Specific amounts may include about 1-150,about 1-10, about 10-25, about 25-50, about 50-75, about 75-100, orabout 100-150 mg tannin/kg body weight per day.

The tannin may be administered via a tube into the gastrointestinaltract that extends at least through the mouth or extends at leastthrough the mouth and throat.

The subject to which the tannin is administered is preferably a mammal,such as a primate, human, rodent, canine, feline, bovine, ovine, equine,swine, caprine, bovine and the like.

The tannin is preferably administered to a subject receiving simplifiednutrition, such as a simplified nutrition composition. The subject maybe receiving such simplified nutrition during enteral or parenteralnutrition therapy. The phrase “administering to a subject receivingsimplified nutrition” and variants thereof, used with respect toadministering tannin, refers to administering the tannin before, during,and/or after delivery of the simplified nutrition. If the tannin isadministered before delivery of the simplified nutrition, theadministering preferably occurs within a timeframe such that any effectsof the tannin administration overlaps with delivery of the simplifiednutrition, such that any deleterious effects of the simplified nutritionare prevented or minimized. If the tannin is administered after deliveryof the simplified nutrition, the administering preferably occurs withina timeframe such that any deleterious effects that do occur as a resultof the simplified nutrition delivery are capable of being ameliorated.As used herein, “delivery” refers to the actual act of delivering anutrient composition to the subject, either orally (as a solid or liquidfoodstuff), enterally (via a tube through, e.g., the nasogastric systemor otherwise), or parenterally (via injection or infusion into thebloodstream). The simplified nutrition composition may be administeredvia a tube into the gastrointestinal tract that extends at least throughthe mouth or that extends at least through the mouth and throat.

The administered simplified nutrition composition may comprise anyenteral or parenteral nutrition composition known or used in the art.Generally, the administered simplified nutrition composition maycomprise one or more of a nitrogen source, a carbohydrate source, and alipid source.

The nitrogen source is preferably in the form of individual amino acidsand/or polypeptides. Exemplary nitrogen sources include casein,caseinates, soy protein, whey protein, lactalbumin, milk proteinconcentrate, hydrolyzed casein, hydrolyzed soy protein, hydrolyzed wheyprotein, hydrolyzed lactalbumin, hydrolyzed milk protein concentrateand/or crystalline L-amino acids.

In some simplified nutrition compositions, the nitrogen source isexclusively comprised of individual amino acids or primarily comprisedof individual amino acids and polypeptides having a short chain length.In various versions of the invention, for example, at least about 1% bymass, at least about 5% by mass, at least about 10% by mass, at leastabout 20% by mass, at least about 30% by mass, at least about 40% bymass, at least about 50% by mass, at least about 60% by mass, at leastabout 70% by mass, at least about 80% by mass, at least about 90% bymass, at least about 95% by mass, or at least about 99% by mass of thenitrogen source is comprised of either individual amino acids orindividual amino acids in combination with polypeptides having, onaverage, a short chain length. As used herein, “short chain length” mayrefer to a chain length less than about 50 residues, less than about 40residues, less than about 30 residues, less than about 20 residues, lessthan about 10 residues, less than about 5 residues, or less than about 3residues.

If present in the simplified nutrition composition, the nitrogen sourcemay comprise up to about 99% of the caloric content of the composition,such as about 1-99% of the caloric content of the composition, about10-70% of the caloric content of the composition, or about 15-60% of thecaloric content of the composition. Some simplified nutritioncompositions may be substantially devoid of a nitrogen source, such asamino acids or polypeptides.

The carbohydrate source may comprise any combination of simplecarbohydrates (i.e., monosaccharides and disaccharides) and complexcarbohydrates. Exemplary carbohydrate sources include glucose, sucrose,fructose, dextrose, glucose polymers, starches such as corn starch orhydrolyzed cornstarch, dextrin, maltodextrin, and sugar alcohols.

Some simplified nutrition compositions have a high proportion of simplecarbohydrates. In various versions of the invention, for example, atleast about 1% by mass, at least about 5% by mass, at least about 10% bymass, at least about 20% by mass, at least about 30% by mass, at leastabout 40% by mass, at least about 50% by mass, at least about 60% bymass, at least about 70% by mass, at least about 80% by mass, at leastabout 90% by mass, at least about 95% by mass, or at least about 99% bymass of the carbohydrate source is comprised of monosaccharides or acombination of monosaccharides and disaccharides.

If present in the simplified nutrition composition, the carbohydratesource may comprise up to about 99% of the caloric content of thecomposition, such as about 1-99% of the caloric content of thecomposition, about 10-80% of the caloric content of the composition, orabout 20-70% of the caloric content of the composition. Somecompositions of the invention may be substantially devoid of acarbohydrate source.

The lipid source preferably comprises free fatty acids and/or glycerides(i.e., monoglycerides, diglycerides, and triglycerides). Exemplary lipidsources include fatty acid esters, fish oil, medium chain triglycerides,safflower oil, sardine oil, soybean oil, soy lecithin, structuredlipids, borage oil, canola oil, corn oil, fish oil, high oleic sunfloweroil, medium chain triglycerides, menhaden oil, mono- and diglycerides,palm kernel oil, safflower oil, soybean oil, soy lecithin, omega-3 fattyacids, and omega-6 fatty acids.

Some simplified nutrition compositions are low in fat with a smallproportion of calories being derived from long-chain fatty acid lipids.In various versions of the invention, for example, the lipid sourcecomprises less than about 1% by mass, less than about 5% by mass, lessthan about 10% by mass, less than about 20% by mass, less than about 30%by mass, less than about 40% by mass, less than about 50% by mass lessthan about 60% by mass, less than about 70% by mass, less than about 80%by mass, less than about 90% by mass, less than about 95% by mass, orless than about 99% by mass of long-chain fatty acid lipids. In someversions, the lipid source is substantially devoid of long-chain fattyacid lipids.

Some simplified nutrition compositions comprise medium-chain fatty acidlipids instead of long-chain fatty acid lipids. Accordingly, in variousversions of the invention, at least about 1% by mass, at least about 5%by mass, at least about 10% by mass, at least about 20% by mass, atleast about 30% by mass, at least about 40% by mass, at least about 50%by mass, at least about 60% by mass, at least about 70% by mass, atleast about 80% by mass, at least about 90% by mass, at least about 95%by mass, or at least about 99% by mass of the lipid source is comprisedof short- or medium-chain fatty acid lipids.

If present in the simplified nutrition composition, the lipid source maycomprise up to about 99% of the caloric content of the composition, suchas about 1-99% of the caloric content of the composition, about 10-70%of the caloric content of the composition, or about 20-60% of thecaloric content of the composition. Some simplified nutritioncompositions may be substantially devoid of a lipid source.

The various proportions of the nitrogen source, the carbohydrate source,and the lipid source may be adjusted based on the nutritional or medicalneeds of the subject. For example, some compositions may comprise about40% of caloric content as a carbohydrate source, about 20% of caloriccontent as a nitrogen source, and about 40% of caloric content as alipid source. Others may comprise about 60% of caloric content as acarbohydrate source, about 30% of caloric content as a nitrogen source,and about 20% of caloric content as a lipid source. Others may compriseabout 80% of caloric content as a carbohydrate source, about 10% ofcaloric content as a nitrogen source, and about 10% of caloric contentas a lipid source. Certain simplified nutrition compositions can containabout 17-25% of the total calories from a nitrogen source, about 35-50%of total calories from a nitrogen source, and about 25-48% of totalcalories from a carbohydrate source.

The caloric density of the simplified nutrition composition may have anycaloric content. In some versions, the caloric content is preferably atleast about 1 Kcal/mL, at least about 1.2 Kcal/mL, at least about 1.3Kcal/mL, at least about 1.5 Kcal/mL, or about 1.3-1.5 Kcal/mL.

The administered simplified nutrition composition may comprise more thanabout 10%, more than about 20%, more than about 30%, more than about30%, more than about 40%, more than about 50%, more than about 60%, morethan about 70%, more than about 80%, more than about 90%, more thanabout 95%, more than about 99%, or about 100% of the subject's dailycaloric intake. Furthermore, the subject may receive the simplifiednutrition composition at such levels over a period of at least about oneday, at least about two days, at least about three days, at least about4 days, at least about 5 days, at least about 6 days, at least about 7days, at least about two weeks, at least about three weeks, or longer.

In preferred versions of the invention, the tannin is administered to asubject receiving a simplified nutrition composition that, in theabsence of tannin, results in a reduction in ileal IL-4, ileal IL-13,goblet cell density, goblet cell size, luminal MUC2 concentration,Peyer's patch lymphocytes, STAT6 phosphorylation, polymericimmunoglobulin receptor (pIgR), luminal secretory immunoglobulin-A(sIgA), and/or gut microbiota diversity in the subject. Accordingly, thetannins of the present invention are preferably administered in anamount and within a timeframe effective to counteract, minimize, orprevent such reductions. Thus, the tannins of the present invention arepreferably administered in an amount and within a timeframe effective toprovide a relative increase in ileal IL-4, ileal IL-13, goblet celldensity, goblet cell size, luminal MUC2 concentration, Peyer's patchlymphocytes, STAT6 phosphorylation, polymeric immunoglobulin receptor(pIgR), luminal secretory immunoglobulin-A (sIgA), and/or gut microbiotadiversity in the subject compared to the same subject not administeredthe tannins.

Additional considerations in the administration of the tannins aredescribed by Ofman et al. Clinical economics review: nutritionalsupport. Ailment Pharmacol Ther 1997, 11:453-471; and McClave et al.Journal of Parenteral and Enteral Nutrition, 33(3):277-316. Additionalcomponents, methods, and data are described by the doctoral thesis ofJoseph Francis Pierre, “Parenteral and Elemental Enteral NutritionDecreases Intestine Mucosal Immunity, Which is Partially Restored byDietary Proanthocyanidins and IL-25,” from University ofWisconsin—Madison; Pierre et al. Journal of Parenteral and EnteralNutrition, 2013, 37(3):401-409; and Pierre J F, Heneghan A F, FelicianoR P, Shanmuganayagam D, Krueger C G, Reed J D, Kudsk K A. CranberryProanthocyanidins Improve Intestinal sIgA During Elemental EnteralNutrition. Journal of Parenteral and Enteral Nutrition, 2013.

EXAMPLES

Abbreviations: DP, degree of polymerization; EEN, elemental enteralnutrition; GAE, gallic acid equivalents; GC, goblet cells; GALT,gut-associated lymphoid tissue; ICR, Institute of Cancer Research;JAK-STAT, Janus kinase/signal transducer and activator of transcription;MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flightmass spectrometry; MUC2, mucin2; PAC, proanthocyanidins; PAS, periodicacid-schiff; PN, parenteral nutrition; PP, Peyer's Patch; sIgA,secretory immunoglobulin-A; pIgR, polymeric immunoglobulin receptor;Th-2, T-helper 2 lymphocytes.

Example 1 Cranberry Proanthocyanidins Attenuate the Effects of ElementalEnteral Nutrition on Size, Density and Function of Intestinal GobletCells in Mice

Lamina propria IL-4 and IL-13 stimulate goblet cell (GC) proliferationand production of mucin 2 (MUC2) by goblet cells, which protectsintestinal mucosa from bacterial pathogens. Elemental enteral nutrition(EEN) reduces IL-4 and IL-13 and impairs gut barrier function. Becausecranberry proanthocyanidins (PAC) were previously shown to stimulateglycoprotein secretion in the oral cavity, we examined the effect of theaddition of PAC to EEN on GC, ileal cytokines, and luminal MUC2. MaleICR mice (14 mice/group) were administered oral chow (e.g., LaboratoryRodent Diet 5001; www.labdiet.com), intragastric EEN, or intragastricEEN+PAC (8 mg (EEN+lowPAC), 50 mg (EEN+midPAC) or 100 mg (EEN+highPAC)of PAC/kg body weight) for 5 days, starting 2 days after intragastriccannulation. Ileal tissue was analyzed for IL-4, IL-13 and GC histology.Intestinal wash fluid was analyzed for MUC2. IL-4 and IL-13 weresignificantly lower in EEN than in the chow group. These effects weresignificantly attenuated in EEN+midPAC and EEN+highPAC groups. Thedensity of GC (GC/villi), GC size and luminal MUC2 were also lower inEEN than in chow group. These effects were also significantly attenuatedby the addition of PAC to EEN (EEN+midPAC and EEN+highPAC). This studyindicates that the addition of cranberry PAC to EEN may counteract theimpairment of barrier function of the intestinal mucosa produced by EENby inhibiting changes in ileal IL-4 and IL-13 levels, GC density andsize, and the secretion of intestinal MUC2.

This example demonstrates that the addition of physiologically relevantdoses of cranberry PAC to EEN attenuates the negative effects of EEN onintestinal barrier function as determined by changes in IL-4 and IL-13,GC density and size, and luminal MUC2.

Materials and Methods.

PAC Preparation.

Non-depectinized cranberry presscake was ground with liquid nitrogen and100 g were extracted with 400 mL of 70% acetone (Fisher Scientific, FairLawn, N.J.). Samples were sonicated and centrifuged at 400×g and 15° C.,for 10 minutes. The extraction was repeated twice. Acetone was removedby evaporation at 35° C. and the aqueous suspension was solubilized inethanol (Decon Labs Inc., King of Prussia, Pa.), followed bycentrifugation at 13,416×g, for 10 minutes at 0° C. to eliminate ethanolinsoluble material. Cranberry presscake crude extract was loaded on aSephadex LH-20™ (GE Healthcare, Uppsala, Sweden) column that waspreviously swollen in water and equilibrated with ethanol for 45 minutesat 4 mL/min. Isolation of PAC was accomplished by sequential elutionwith ethanol, ethanol/methanol (1:1) and 80% acetone. Acetone in thelast fraction that contained PAC was removed by evaporation under vacuumand the PAC was re-solubilized in methanol (Fisher Scientific, FairLawn, N.J.). The total phenolic content of the PAC fraction wasdetermined by the modified Folin-Ciocalteu method (Singleton and Rossi,Amer. J. Enology and Viticulture, 1965; 16:144-58) and reported asgallic acid equivalents (GAE).

PAC Characterization by HPLC and MALDI-TOF MS.

The cranberry presscake PAC sample was diluted tenfold to reduce theamount of methanol to 10%. One hundred microliters were injected onto aWaters Spherisorb® 10 μm ODS2 RP-18 column (4.6×250 cm). The solventsfor elution were trifluoroacetic acid/water (0.1%; solvent A) andmethanol (solvent B). A step gradient was used, starting with 90%solvent A and 10% solvent B for 10 minutes, isocratic 72% A and 28% Bbetween 10 and 25 minutes; a linear gradient from 28% to 55% B between25 and 45 minutes; 45-50 minutes, a linear gradient from 55 to 99% Bbetween 50 and 55 minutes, and isocratic 1% A and 99% B between 55 and60 minutes. The HPLC system consisted of a Waters automated gradientcontroller, two Waters 501 HPLC pumps, and a Rheodyne 7125 manualinjector. The flow rate was maintained at 2 mL/min, and the elution wasmonitored by a Waters 996 diode array detector using Waters Millenniumsoftware for collecting and analyzing three-dimensional chromatograms.

PAC characterization by matrix-assisted laser desorption/ionizationtime-of-flight MS (MALDI-TOF MS). The cranberry presscake PAC sample wasmixed with 2,5-dihydroxybenzoic acid (Aldrich, Milwaukee, Wis.) (50mg/mL in 100% ethanol) and the mixture was applied (1 μL) onto aMALDI-TOF MS stainless steel target and dried at room temperature. Massspectra were collected on a Bruker Reflex II MALDI-TOF-MS (Billerica,Mass.) equipped with delayed extraction and a N2 laser (337 nm) in orderto characterize the range in degree of polymerization and nature ofinterflavan bonds in the cranberry PAC. All preparations were analyzedin the positive ion linear and reflectron mode to detect [M+Na]⁺ and[M+K]⁺ molecular ions. MALDI-TOF MS is ideally suited for characterizingPAC because, unlike electrospray ionization in which multiple chargemolecular ions create very complex spectral peaks that are oftendifficult to interpret, this mass spectral technique produces only asingly charged molecular ion for each parent molecule (Reed, Krueger,and Vestling, Phytochemistry, 2005; 66:2248-63).

Animals.

Male Institute of Cancer Research (ICR) mice were purchased throughHarlan (Indianapolis, Ind.). The mice were acclimatized for one week ina temperature and humidity controlled environment with a 12 h/12 hlight/dark cycle. The mice were housed 5 per microisolater-top cages andfed ad libitum chow (Rodent Diet 5001, LabDiet, PMI NutritionInternational, St. Louis, Mo.) and water for 1 week prior to initiationof study protocol. Once entering study protocol, the mice were housedindividually in metal wire-bottomed cages to prevent coprophagia andingestion of bedding.

Experimental Design.

Seventy male ICR mice (6 to 8 wk old) were anesthetized withintraperitoneal administration of ketamine (100 mg/kg) and acepromazine(10 mg/kg). Gastrostomy was then performed on each mouse and thecatheter was tunneled subcutaneously from the gastrostomy site, over theback, finally exiting mid-tail. The mice were partially restrained bythe tail for the remainder of the study to protect the catheter duringinfusions (Sitren et al., J Parenter Enteral Nutr. 1983; 7:582-6). Thispartial restraint technique does not induce significant stress in themice. The catheterized mice were connected to infusion pumps and allowedto recover for 48 hours while receiving 4 mL/d of saline (0.9%) via thecatheter. The mice also received ad libitum chow (Rodent Diet 5001,LabDiet) and water.

Following the recovery period, the mice were randomized (n=14/dietgroup) to receive oral chow, intragastric EEN or intragastric EEN+PAC [8mg (EEN+lowPAC), 50 mg (EEN+midPAC) or 100 mg (EEN+highPAC) GAE ofPAC/kg body weight]. The chow-fed mice were given ad libitum chow andwater, and continued to receive 0.9% saline at 4 mL/d via theintragastric catheter. EEN and EEN+PAC fed mice received solution at 4mL/d (day 1), 7 mL/d (day 2) and 10 mL/d (days 3-5) as well as adlibitum water throughout the study. The EEN solution (Table 1-1)included 6% amino acids, 35.6% dextrose, electrolytes, andmultivitamins, with a non-protein calorie to nitrogen ratio of 126.1(527.0 kJ/g nitrogen). This value meets the calculated nutrientrequirements of mice weighing 25 to 30 g.

TABLE 1-1 Formulation of EEN Solution. Component Amount (per 1 L)Dextrose 356.0 g Amino acids (Clinisol) 60.0 g Sodium chloride 32.0 mEqSodium phosphate 36 mmol Potassium chloride 16 mEq Calcium gluconate37.5 mEq Potassium acetate 44.0 mEq Magnesium sulfate 8.0 mEq Manganese0.8 mg Copper 0.5 μg Zinc 2.0 mg Vitamin C 200 mg Vitamin A 3300 IUVitamin D₃ 200 IU Thiamine 6 mg Riboflavin 3.6 mg Pyridoxine HCl 6 mgNiacinamide 40 mg Folic acid 600 mcg Biotin 60 mcg Cyanocobalamin 5 mcgVitamin E (dl-α-tocopheryl acetate) 10 IU Vitamin K₁ 150 mcgDexpanthenol 15 mg

After 5 d of feeding (7 days post-catheterization), mice wereanesthetized as before, and exsanguinated via left axillary arterytransection. The small intestine from each mouse was removed and thelumen rinsed with 20 mL HBSS (Bio Whittaker, Walkersville, Md.). Theluminal rinse was centrifuged at 2,000×g for 10 minutes, and supernatantwas aliquoted and frozen at −80° C. for MUC2 analysis. Ileal tissuesamples were obtained from a 3 cm segment of ileum that excluded Peyer'spatches. Samples for ileal IL-4 and IL-13 determination wereflash-frozen in liquid N2 and stored at −80° C. until subsequentanalysis, while samples for GC analysis were fixed in 4%paraformaldehyde overnight, transferred to 70% ethanol, and stored at 4°C. until subsequent histology.

Analysis of Ileal IL 4 and IL 13.

The flash-frozen small intestine segment from each animal washomogenized in RIPA lysis buffer (Upstate, Lake Placid, N.Y.) containing1% protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, Mo.).The homogenate was kept on ice for 30 minutes prior to centrifugation at16,000×g for 10 minutes at 4° C. The supernatant was then stored at −20°C. until analysis. Prior to storage, the protein concentration of thesupernatant was determined by the Bradford method using BSA as astandard.

Concentrations of IL-4 and IL-13 were determined in the supernatantusing solid phase sandwich ELISA kits (BD Biosciences, San Diego,Calif.), according to manufacturer's instructions. The absorbance at 450nm was determined using a Vmax Kinetic Microplate Reader (MolecularDevices, Sunnyvale, Calif.). The IL-4 and IL-13 concentrations in thesamples were determined by using a 4-parameter logistic fit standardcurve (SOFTmax PRO software; Molecular Devices; Sunnyvale, Calif.) andnormalized to total tissue protein content.

Analysis of Luminal MUC2.

The proteins in the intestinal wash fluid (4 μL) from each animal wereseparated by 10% agarose gel by electrophoresis at 150V for 80 minutesat room temperature (˜23° C.). The resolved proteins were transferred toa polyvinylidene fluoride membrane using tris-glycine buffer containing20% methanol at 80V for 60 minutes at 4° C. The membrane was blockedwith 5% nonfat dry milk prepared in Tris buffered saline containingTween (0.05%) for 1 hour at room temperature with constant agitation.Then, the membrane was incubated with mouse anti-human MUC2 (ab-11197,Abcam Inc., Cambridge, Mass.) primary antibody (diluted 1:2500)overnight at 4° C. with constant agitation. The membrane was washed andincubated with stabilized goat anti-mouse IgG-HRP conjugate (sc-2005,Santa Cruz Biotechnology, CA) secondary antibody (diluted 1:20,000) for1 hour at room temperature with constant agitation. After washing, themembrane was incubated with HRP substrate (Super Signal West Femtosubstrate; Pierce, Rockford, Ill.) for 5 minutes and the protein ofinterest (MUC2) was detected using photographic film. The relativeintensity of MUC2 for each sample was determined using NIH ImageJsoftware (version 1.43, http://rsbweb.nih.gov/ij/) using internalcontrols normalize the densitometry analysis across multiple film.

Analysis of GC Density and Size.

The fixed ileal tissue sections were processed (Tissue-Tek V.I.P, SakuraFinetek, Torrance, Calif.), and embedded in paraffin. The embeddedtissue was cut (5 μm thick), deparaffinized, rehydrated through gradedethanol washes (100% ethanol×2, 95% ethanol×2, 70% ethanol×1, 2 minuteseach) and placed into distilled H2O, Samples were stained with periodicacid-schiff (PAS) and counterstained with hematoxylin. GC density(GC/villi) was determined by counting taking the average number of GCpresent in 15 individual villi. GC size (μm2) was obtained by imagingtissue sections and analyzing individual GC with NIH ImageJ software(version 1.43, http://rsbweb.nih.gov/ij/).

Statistical Analysis.

The number of samples used from each diet group for each analysis isindicated in the corresponding figures. The significance of thedifferences between each diet group for each measured parameter wasassessed by one-way ANOVA and the Fisher protected least significantdifference (PLSD) post hoc test corrected for multiple comparisons(Statview 5.0.1, SAS, Cary, N.C.). Statistical significance was acceptedat α=0.05. Values are presented as mean±SEM.

Results.

PAC Characterization by HPLC and MALDI-TOF MS.

The cranberry presscake PAC eluted as two unresolved humps that had peakabsorbance at 280 nm and minor absorbance at 520 nm due to the presenceof covalently linked anthocyanin-proanthocyanidin pigments. No peakswith an absorbance max that is typical of the other classes of cranberrypolyphenolic compounds (anthocyanins, hydroxycinnamic acids, andflavonols) were observed. The poorly resolved chromatogram at 280 nmreflects the large structural heterogeneity of cranberry presscake PAC(Reed, Krueger, and Vestling, Phytochemistry, 2005; 66:2248-63).

Reflectron mode MALDI-TOF MS showed masses that correspond to PAC withat least 1A-type interflavan bond in trimers to undecamers (Table 1-2).MALDI-TOF MS linear mode spectra had m/z peaks that correspond tocranberry presscake PAC with a range of 3 to 23 DP. The spectra alsocontained m/z peaks that correspond to covalently linkedanthocyanin-proanthocyanidin molecules, ranging from monomers toheptamers (data not shown).

TABLE 1-2 Calculated and observed m/z for PAC isolated from cranberrypresscake. All B-type bonds One A-type bond Two A-type bonds ThreeA-type bonds Calcd. Obs. Calcd. Obs. Calcd. Obs. Calcd. Obs. Trimers889.2 889.2 887.2 887.2 885.2 885.2 Tetramers 1177.2 1177.5 1175.21175.5 1173.2 1173.5 1171.2 1171.4 Pentamers 1465.3 1465.8 1463.3 1463.81461.3 1461.8 1459.3 1459.8 Hexamers 1753.3 1754.0 1751.3 1752.0 1749.31749.9 1747.3 1748.2 Heptamers 2041.4 2042.3 2039.4 2040.3 2037.4 2038.32035.4 2036.4 Octamers 2329.5 2330.4 2327.4 2328.7 2325.4 2326.6 2323.42324.7 Nonamers 2617.6 2618.9 2615.6 2617.0 2613.5 2615.0 2611.5 2613.2Decamers 2905.6 2906.2 2903.5 2904.1 2901.5 2902.0 2899.5 2900.7Undecamers 3191.7 3192.4 3189.7 3190.3 3187.6 3188.9The observed masses were determined by MALDI-TOF MS and the calculatedmasses were based on m/z=290+288d−2A+c, where 290 represents themolecular weight of the terminal catechin/epicatechin unit, d is thedegree of polymerization, A is the number of A-type interflavan bondsand c is the molecular weight of sodium cations. Blank spaces indicatethat a mass was not observed (signal to noise ratio <3.0).

Analysis of Ileal IL-4 and IL-13.

IL-4 level in the ileal tissue of the EEN group (4.5±0.4 ρg/mg totalprotein) was significantly lower than in the chow group (6.2±0.4,P<0.05) (FIG. 1). IL-4 level in the EEN+highPAC group (7.0±0.5) wassignificantly higher than in the EEN group (P<0.005), while neither thelevels in EEN+lowPAC (5.0±0.7) nor EEN+midPAC (5.8±0.6) groupssignificantly differed from that in the EEN group.

Although IL-13 in the ileal tissue was significantly lower in the EENgroup (7.5±1.0 ρg/mg total protein) than in the chow group (11.4±1.9)(FIG. 2), the difference did not reach statistical significance(P=0.08). IL-13 in the EEN+midPAC (11.8±1.3, P<0.05) and EEN+highPAC(13.9±2.1, P<0.01) were significantly higher than in the EEN group.

Analysis of GC Density and Size.

The density of GC (FIG. 3) in the EEN group (8.0±0.3 GC/villi) wassignificantly lower than in the chow group (9.7±0.7, P<0.005). The GCdensity in the EEN+lowPAC (9.2±0.4, P<0.05), EEN+midPAC (9.7±0.4,P<0.01), and EEN+highPAC (10.4±0.3, P<0.0001) groups were significantlyhigher than in the EEN group. The GC density in the EEN+highPAC groupwas also significantly greater than in the EEN+lowPAC group (P<0.05).

Although the GC size in the EEN group (9.0±0.6 μm2) was smaller than inthe chow group (10.4±0.4), the difference did not reach statisticalsignificance (P=0.23) (FIG. 4). The GC sizes in the EEN+lowPAC(12.0±0.9, P<0.01), EEN+midPAC (12.4±0.9, P<0.01), and EEN+highPAC(11.8±0.8, P<0.05) groups were significantly greater than in the EENgroup.

Analysis of Luminal MUC2.

The relative luminal MUC2 within the small intestine in the EEN (75±11%of chow group, P<0.05) and EEN+lowPAC (65±9% of chow group, P<0.05)groups were significantly lower in the chow group (FIG. 5). However, therelative luminal MUC2 in the EEN+midPAC (99±14% of chow group, P<0.05)and EEN+highPAC (115±16% of chow group, P<0.005) groups weresignificantly higher than in the EEN group.

Discussion.

A major finding of this example is that the addition of cranberry PAC toEEN solution attenuates the effects of EEN on ileal tissue IL-4 andIL-13 levels, GC density and size, and the secretion of intestinal MUC2.Reduced ileal tissue IL-4 and IL-13 levels, GC density and size, andsecretion of intestinal MUC2 contributes to the impairment of themucosal barrier integrity observed in conjunction with EEN therapy.

PAC are complex oligomeric polyphenolic compounds widely distributed infruits, including grapes, cranberries, and apples, and other foods andbeverages such as chocolate and wine. Epidemiological studies suggestPAC may have beneficial health effects. However, PAC are minimallyabsorbed across the enterocyte layer due to non-hydrolyzable bondsbetween flavan-3-ol monomeric units and their ability to complex bothdietary and endogenous proteins. Further, PAC oligomers range in DP from3 to 30, or more, and therefore have higher molecular weight than othercommon plant polyphenols. Consequentially, greater than 95% of PACremain in the intestinal lumen during transit through thegastrointestinal tract.

While PAC are poorly absorbed, they can provide several beneficialeffects. PAC can exert antioxidant and non-specific antimicrobialfunctions within the gut, and the addition of dietary PAC can palliatechemically-induced colitis. PAC can also complex salivary glycoproteinswhen ingested, a process that causes the astringency of many fruits andbeverages. Astringency occurs when PAC crosslink and precipitatesalivary glycoproteins. PAC with higher DP have greater effects oncrosslinking and precipitation. Several biological effects occur inresponse to astringency including increased salivary excretion,hypertrophy of the parotid gland, and shift in salivary composition toproline rich proteins. In response to PAC within the intestine,intra-epithelial γδT lymphocytes activate and proliferate.Interestingly, the level of γδT cell response also increases withgreater DP of PAC. This indicates that PAC can improve mucosal barrierphysiology and immunity, and that an increased DP of PAC can provideadditional benefits.

The cranberry presscake PAC was first analyzed by HPLC to confirm thatthe preparation did not contain other classes of cranberry phenoliccompounds. A MALDI-TOF MS technique that is capable of determining thestructural complexity of PAC was used to estimate the range in DP andelucidate PAC structural features (Reed, Krueger, and Vestling,Phytochemistry, 2005; 66:2248-63; Hanton, Chem. Rev. 2001; 101:527-69;Krueger, Vestling, Reed, J. Agric. Food Chem. 2003; 51:538-43). Bothlinear and reflectron modes were used in this work because they providecomplimentary information. High resolution was achieved in thereflectron mode while high sensitivity was obtained in the linear mode,particularly at high molecular weight samples.

The cranberry presscake PAC oligomers detected in this example inreflectron mode are consistent with other work in which PAC ranging fromtetramers to tridecamers were detected (White et al., J. Agric. FoodChem. 2010; 58:4030-6). Higher resolution of reflectron mode spectraallowed detection of overlapping isotope patterns due to the presence ofPAC with 1, 2 and 3A-type linkages that are 2 amu apart (Table 1-2), aspreviously reported (Neto et al., J. Sci. Food Agric. 2006; 86:18-25;Porter et al., J. Sci. Food Agric. 2001; 81:1306-13). Linear modeMALDI-TOF MS detected oligomers with a DP of 23 flavan units. Althoughthe cranberry presscake PAC preparation is a complex mixture of closelyrelated oligomers, detailed analysis by HPLC and MALDI-TOF MS allowedfor the characterization and reliable reproduction of chromatographicfractions for inclusion in experimental diets.

In this example, investigations evaluated the effects of addition ofcranberry PAC to EEN solution on ileal tissue IL-4 and IL-13 levels, GCdensity and size, and the secretion of the primary glycoprotein MUC2,and explored the effect of physiological doses of PAC on theseparameters. A chemically-defined EEN solution administered via agastrostomy tube was used as a model of an elemental enteral diet (Li etal., J. Trauma 1995; 39:44-51). The EEN administration results inreproducible effects on intestinal (and respiratory) mucosal immunityallowing examination of changes induced with PAC. While the lower GCsize observed with EEN compared to chow were not statisticallysignificant level, the data demonstrate that EEN produces significantlylower density of ileal GC compared to chow feeding. Since GCdifferentiate, migrate up the villi, and slough off every 3-5 days,these findings indicate that reduced dietary complexity alters the rateof cellular differentiation of progenitor crypt stem cells to GC, likelyvia changes in Th-2 type cytokines observed with EEN.

Goblet cells normally undergo hypertrophy and hyperplasia in response toIL-4 and IL-13, which act through the IL-4 receptor α and IL-13 receptorα1, respectively. The data presented herein shows that EEN lowers ilealIL-4 and IL-13 levels. Simultaneously, EEN significantly decreases theconcentration of MUC2 within the lumen. Functionally, MUC2 forms theviscous mucin layer that overlays the intestinal surface, allowingsmooth passage of digesta. From an immunological stand point, secretedantimicrobial proteins and peptides from Paneth cells as well assecretory IgA (sIgA) localize and are concentrated in this layer. Thesemucin glycoproteins also provide endogenous flora under a consistentnutrient source. The observed decrease in luminal MUC2 likely increasessusceptibility to bacterial opportunistic pathogens or intestinalinflammation, which effect is supported by findings of others showingthat MUC2−/− mice are at increased risk for spontaneous colitis(Bergstrom et al., PLoS Pathog. 2010; 6:e1000902).

Overall, this example shows that reduced enteral stimulation results inthe impairment of mucosal integrity and gut barrier function through thereduction in the mucin component. This example demonstrates that theadministration of EEN produces lower levels of Th2 stimulatingcytokines, IL-4 and IL-13, lower GC density and size, and lower luminalMUC2 levels in the ileum. The addition of cranberry PAC to this diet, atphysiologic doses, attenuates these changes and normalizes mucosalintegrity. This indicates that a non-nutritional dietary component suchas PAC can influence health without being absorbed from thegastrointestinal tract, thereby providing a significant benefit whenused in conjunction with a restricted diet such as elemental enteralnutrition.

Example 2 Cranberry Proanthocyanidins Improve Intestinal sIgA DuringElemental Enteral Nutrition

Elemental enteral nutrition (EEN) decreases gut-associated lymphoidtissue (GALT) function, including fewer Peyer's patch lymphocytes, lowerlevels of the tissue Th2 cytokines and mucosal transport proteinpolymeric immunoglobulin receptor (pIgR), leading to lower luminal sIgAlevels. Example 1 above demonstrates that cranberry proanthocyanidins(PAC) maintain the Th2 cytokine IL-4 when added to EEN. This exampleshows that the addition of PAC to EEN normalizes other GALT parametersand maintains luminal levels of sIgA.

Briefly, ICR mice were randomized (12/group) to receive Chow, EEN, orEEN+PAC (100 mg/kg body weight) for 5 days, starting 2 days afterintra-gastric cannulation. Ileum tissue was collected to measure IL-4 byELISA, pIgR by western blot, and phosphorylated STAT6 by microarray.Intestinal wash fluid was collected to measure sIgA by western blot.Compared with Chow, EEN significantly decreased tissue IL-4,phosphorylated STAT6, and pIgR. The addition of PAC to EEN preventedthese alterations. Compared with Chow, EEN resulted in significantlylower levels of luminal sIgA. The addition of PAC to EEN increasedluminal sIgA levels compared to EEN alone. Therefore, the addition ofPAC to EEN can support GALT function and maintain intestinal sIgA levelscompared with EEN alimentation alone.

Decreased dietary bulk and complexity provided with EEN attenuatesmucosal agitation and painful symptoms. Unfortunately, reduced dietarycomplexity, such as provided with EEN or parenteral nutrition (PN),alters the structure and function of the gut-associated lymphoid tissue(GALT). Ultimately, reduced dietary complexity manifests as decreasedsecretory immunoglobulin-A (sIgA) in the gut lumen compared to enteralfeeds. sIgA is the primary protective compound of acquired immunitysecreted by the host mucosa, which among other notable functions excludeenteric bacteria from attachment to the host. EEN also results inincreased bacterial translocation and decreased microbiome diversity. Toaddress EEN-induced susceptibilities, various interventions have beeninvestigated to provide anti-inflammatory and protective effects in thegut. An established feeding model employing intra-gastric administeredEEN results in the reproducible loss of intestinal (and respiratory)sIgA. This example evaluated whether a class of natural compoundsisolated from cranberries, proanthocyanidins (PAC), support mucosalprotection by stimulating luminal sIgA levels when added to EEN.

Reduced luminal sIgA levels following EEN or parenteral nutrition ismultifactorial, including fewer lymphocyte numbers in both Peyer'spatches (PP) and lamina propria compartments; suppressed T helper 2(Th2) cytokines, IL-4 and IL-10, in the lamina propria; and reducedexpression of mucosal pIgR, which is the primary transport protein forsIgA. Expression of pIgR is regulated in part through the Januskinase/signal transducer and activator of transcription (JAK/STAT)pathway, a cytokine signaling cascade used to transduce a wide array ofcellular events. IL-4 binds the IL-4 receptor-α inducing intracellularSTAT6-phosphorylation, dimerization, and migration into the cell nucleustargeting transcription products, including pIgR. EEN decreases sIgAlevels and PAC support intestinal Th2 cytokines. Additionally, it wasfound that the addition of physiological PAC doses to EEN supports GALTfunction and luminal sIgA compared with EEN alimentation alone.

PAC preparation and characterization was carried out as described inExample 1. Mice were obtained and acclimatized as described in Example1.

Experimental Design.

Male ICR mice, ages 6 to 8 weeks, were randomized to Chow with a gastriccatheter (n=12), intragastric elemental nutrition (EEN) (n=12) viagastrostomy, or EEN+PAC via gastrostomy (100 mg/kg body weight(EEN+PAC)) (n=12). Animals were anesthetized by intraperitonealinjection of ketamine (100 mg/kg) and acepromazine (10 mg/kg). Catheterswere tunneled subcutaneously from the gastrostomy site over the back andexited mid tail. Mice were partially immobilized by tail fixation toprotect the catheter during infusion. This technique does not inducesignificant physical or biochemical stress.

Catherized mice were connected to infusion pumps and allowed recoveryfor 48 hours while receiving 4 mL/day saline (0.9%) and ad libitum chow(Agway Inc., Syracuse, N.Y.) and water. Following the recovery periodexperimental diets were given. Chow mice continued to receive 0.9%saline at 4 mL/day as well as ad libitum chow and water throughout thestudy. The EEN solution includes 6% amino acids, 35.6% dextrose,electrolytes, and multivitamins, with a non-protein calorie/nitrogenratio of 126.1 (527.0 kJ/g Nitrogen). This value meets the calculatednutrient requirements of mice weighing 25 to 30 g. EEN and EEN+PAC fedmice received solution at 4 mL/day (day 1), 7 mL/day (day 2) and 10mL/day (days 3-5) as well as ad libitum water throughout the study.

After 5 days of feeding (7 days post-catherterization), mice wereanesthetized by intraperitoneal injection of ketamine (100 mg/kg) andacepromazine (10 mg/kg), and exsanguinated via left axillary arterytransection. The small intestine was removed and the lumen rinsed with20 mL Hanks Balanced Saline Solution (HBSS, Bio Whittaker, Walkersville,Md.). The luminal rinse was centrifuged at 2,000×g for 10 min andsupernatant aliquots were frozen at −80° C. for sIgA analysis. Tissuesamples were taken by removing a 3 cm segment of ileum excluding PPs. PPlymphocytes were assessed by counting on a hemocytometer. Samples werefrozen in liquid N2 and stored at −80° C. until processing or fixed in4% paraformaldehyde overnight, transferred to 70% ethanol, and stored at4° C. for immunohistochemistry.

Peyer's Patch Lymphocytes.

The Peyer's patch (PP) from the entire length of the SI was removed into1.5 mL tubes of CMF-HBSS. PP were strained through 100-μm mesh with atotal volume of 15 mL CMF-HBSS. The effluent was collected and spun at1700 rpm at 5° C. for 10 min. The supernatant was removed and the pelletresuspended in 15 mL CMF-HBSS; this step was repeated. Cells werecounted on a hemocytometer with trypan blue.

Tissue Cytokine Quantitative Analysis.

The flash-frozen small intestine segment from each animal washomogenized in RIPA lysis buffer (Upstate, Lake Placid, N.Y.) containing1% protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, Mo.).The homogenate was kept on ice for 30 minutes prior to centrifugation at16,000×g for 10 minutes at 4° C. The supernatant was then stored at −20°C. until analysis. Prior to storage, the protein concentration of thesupernatant was determined by the Bradford method using BSA as astandard.

Concentration of IL-4 was determined in the supernatant using solidphase sandwich ELISA kits (BD Biosciences, San Diego, Calif.), accordingto manufacturer's instructions. The absorbance at 450 nm was determinedusing a Vmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale,Calif.). The IL-4 concentrations in the samples were determined by usinga 4-parameter logistic fit standard curve (SOFTmax PRO software;Molecular Devices; Sunnyvale, Calif.) and normalized to total tissueprotein content.

JAK-STAT Profiling by the JAK-STAT Antibody Microarray.

The Phospho Explorer antibody microarray (Full Moon Biosystems Inc.,Sunnyvale, Calif.), contains 42 antibodies. Each of the antibodies hassix replicates that are printed on coated glass microscope slide, alongwith multiple positive and negative controls. The antibody arrayexperiment was performed according to established protocol (Kang et al.,J. Clin. Invest. 2010, 120, 1165-1177). In brief, ileum tissue lysates(n=8/group) were biotinylated with Antibody Array Assay Kit. Theantibody microarray slides were first blocked in a blocking solution for30 minutes at room temperature, rinsed with Milli-Q grade water for 5minutes, and dried with compressed nitrogen. The slides were thenincubated with the biotin-labeled cell lysates (˜80 μg protein) incoupling solution at room temperature for 2 hours. The array slides werewashed 5 times with 1× Wash Solution and rinsed extensively with Milli-Qgrade water before detection of bound biotinylated proteins usingCy3-conjugated streptavidin. The slides were scanned on a GenePix 4000scanner and the images were analyzed with GenePix Pro 6.0 (MolecularDevices, Sunnyvale, Calif.). The fluorescence signal of each antibodywas obtained from the fluorescence intensity of this antibody spot aftersubtraction of the blank signal (spot in the absence of antibody), andthe signal of the phosphorylated protein to GAPDH housekeeping proteinexpression was used.

Analysis of pIgR Expression by Western Blot.

Solubilized protein from small intestinal tissue homogenate wasdenatured at 95° C. for 10 minutes with sodium dodecylsulfate andβ-mercaptoethanol, and 20 μg of protein from each sample was separatedin a denaturing 10% polyacrylamide gel by electrophoresis at 150V for 1hour at room temperature. Proteins were transferred to a PVDF membrane,and western blot was performed as previously described (Sano et al., Am.J. Surg. 2009, 198, 105-109). Densitometric measurements of proteinbands were analyzed and quantified with the NIH Image J software. pIgRstandard (Cat 2800, R&D, Minneapolis, Minn.) was used to comparemultiple gels. The combined value of the 120 kDa and 94 kDa bands wasdetermined for the quantitation of the pIgR protein expression insample.

Analysis of IgA by Western Blot.

Luminal wash IgA was measured by western blot because it was observedthat the addition of PAC to control animal luminal wash samples rapidlydecreases sensitivity and total signal measured by IgA ELISA(unpublished observation), likely through the complexation between PACwith proteins. Four μL of luminal fluid was denatured at 95° C. for 10minutes with sodium dodecylsulfate and β-mercaptoethanol. Proteins wereseparated in a denaturing 10% polyacrylamide gel by electrophoresis at150 V for 1 hour at room temperature and transferred to a polyvinylidenefluoride membrane using tris-glycine buffer plus 20% methanol at 80 Vfor 50 minutes at 4° C. The membrane was blocked with 5% nonfat dry milkprepared in TBS-Tween for 1 hour at room temperature with constantagitation. Membranes were incubated with goat anti-mouse IgA, α-chainspecific (Sigma-Aldrich, St. Louis, Mo.) diluted 1:7,000 for 1 hour atroom temperature with constant agitation. Then, membranes were washedand incubated with stabilized donkey anti-goat IgA-HRP conjugatedsecondary diluted 1:20,000 for 1 hour at room temperature. Afterwashing, membranes were incubated with HRP substrate (Super Signal WestFemto maximum sensitivity substrate; Pierce, Rockford, Ill.) for 5minutes and bands were detected using photographic film. Densitometricmeasurements of immunoglobulin α-chain protein bands (˜55 kDa) wereanalyzed and quantified with the NIH Image J software. IgA heavy chainstandard (M-1421, Sigma-Aldrich) was used to normalize across multiplegels.

Statistical Analysis.

Experimental values were compared using analysis of variance (ANOVA) andFisher protected least significance difference (PLSD) corrected formultiple comparisons, with α=0.05 considered significant (Statview5.0.1, SAS, Cary, N.C.). Numerical results are presented asmean±standard deviation of the mean.

Results.

PAC Characterization by HPLC and MALDI-TOF MS.

The cranberry presscake PAC eluted as two unresolved peaks that hadabsorbance at 280 nm and minor absorbance at 520 nm due to the presenceof covalently linked anthocyanin-proanthocyanidin pigments. No peakswere observed with an absorbance max typical of the other classes ofcranberry polyphenolic compounds (anthocyanins, hydroxycinnamic acids,and flavonols). The poorly resolved chromatogram at 280 nm is due tostructural heterogeneity of cranberry presscake PAC.

Reflectron mode MALDI-TOF MS showed masses that correspond to PAC withat least 1A-type interflavan bond in trimers to undecamers. MALDI-TOF MSlinear mode spectra had m/z peaks that correspond to cranberry presscakePAC with a range of 3 to 23 degrees of polymerization. The spectra alsocontained m/z peaks that correspond to covalently linkedanthocyanin-proanthocyanidin molecules, ranging from monomers toheptamers (data not shown).

Peyer's Patch Lymphocytes.

Compared with Chow (4.533×106±1.226×106 cells), EEN significantlylowered PP lymphocytes (2.428×106±0.574×106 cells, P<0.0001) (FIG. 6).Compared with EEN alone, PP lymphocytes were significantly higher inEEN+PAC (3.957×106±1.291×106 cells, P<0.001). There were no significantdifferences between Chow and EEN+PAC (P=0.19).

Ileum Tissue IL-4.

Compared with Chow (6.5±1.11 pg/mg protein), EEN significantly loweredileum IL-4 (4.15±1.44, P<0.01) (FIG. 7). Compared with EEN alone, ileumIL-4 was significantly higher in EEN+PAC (5.8±2.2, P<0.05). There was nosignificant difference between the level of ileum IL-4 between Chow andEEN+PAC (P=0.42).

Ileum Tissue Phosphorylated STAT6.

Phosphorylated STAT6 (PSTAT6) was measured at two phosphorylation sites,Tyrosine 641 (Tyr641) and Threonine 645 (Thr645), and normalized toGAPDH expression. Compared with Chow (8.66±1.5 PSTAT6 (Tyr641)/GAPDH),PSTAT6 at Tyr641 site was significantly reduced with EEN (6.08±1.3,P<0.001). The addition of PAC to EEN significantly elevated PSTAT6 atTyr 641 (8.11±0.7, P<0.01). There was no difference between Chow andEEN+PAC (P=0.37) (FIG. 8A). Similarly, compared with Chow (8.97±1.6PSTAT6 (Thr645)/GAPDH), PSTAT6 at Thr645 was significantly lower withEEN (6.60±1.0, P<0.01). The addition of PAC to EEN significantlyelevated PSTAT6 at Thr645 (7.99±0.9, P<0.05), however, there was nosignificant difference between Chow and EEN+PAC (P=0.13) (FIG. 8B).

Ileum Tissue pIgR.

EEN (10.23±5.23) lowered tissue pIgR (relative concentration/20 μgprotein) compared with Chow (20.71±7.63, P<0.001) (FIG. 9). PAC+EEN(16.13±5.97, P<0.03) levels of tissue pIgR were significantly higherthan EEN alone. There was no significant difference between Chow andEEN+PAC (P=0.08).

Luminal sIgA.

Compared with Chow (17.62±6.52), the level of luminal sIgA (relativeconcentration/4 μL luminal wash) was significantly lower following EEN(10.33±4.23, P<0.001) (FIG. 10). The addition of PAC to EEN (14.67±5.86,P<0.05) significantly elevated luminal sIgA compared with EEN alone.There was no significant difference between EEN+PAC and Chow (P=0.15).

Discussion.

EEN allows alimentation to patients with contraindication to normalfeeding by administering a liquid diet directly into thegastrointestinal tract. EEN formulas are usually used in clinicalconditions involving intestinal or pancreatic inflammation. Theadministration of a glucose-amino acid infusion (EEN) administered viagastrostomy decreases several aspects of GALT function, including fewerPP and lamina propria lymphocytes; reduced tissue IL-4 and IL-10; pIgR,the sIgA mucosal transport protein; and decreased levels of luminalsIgA. Unfortunately, these changes result in increased susceptibility toinfection and inflammation because sIgA is the primary protectivemolecule of specific (acquired) immunity that is secreted onto mucosalsurfaces. sIgA opsinizes bacteria, preventing their attachment to themucosa, and reduces virulent expression in enteric pathogens. Consistentwith its negative effect on luminal sIgA, EEN also increases mucosalbarrier permeability and decreases microbiome diversity. Because EEN isthe only enteral formula tolerated in certain patients, EEN supplementsthat improve host immune and barrier function are of particular value.In this example, the effect of PAC upon GALT function was investigated.The PAC led to the release of sIgA in the intestinal lumen.

PAC are complex oligomeric polyphenolic compounds distributed in fruits,including grapes, cranberries, and apples, and other foods and beveragessuch as chocolate and wine. PAC do not appear to leave the gut lumen fora variety of reasons, including non-hydrolysable bonds betweenflavan-3-ol monomeric units and their ability to complex both dietaryand endogenous proteins. Further, PAC oligomers range in degree ofpolymerization from 3 to 25+ and therefore have higher molecular weightthan other common plant polyphenols. Due to these characteristics,rodent models demonstrate greater than 95% of PAC remain in theintestinal lumen during transit through the gastrointestinal tract, andingested PAC do not contribute to circulating flavanol levels in humans.PAC can therefore exert beneficial health effects through theirinteraction at the gut mucosa.

The expression of pIgR is regulated through IL-4 stimulation of thenuclear factor STAT-6, a member of the JAK/STAT signaling cascade.STAT-6, in part, regulates luminal sIgA through regulation of themucosal transport protein pIgR. The importance of STAT-6 duringparenteral nutrition with lack of enteral stimulation has beenestablished, showing that lower IL-4 levels correlated levels ofphosphorylated STAT-6, pIgR, and luminal sIgA. Administration ofexogenous cytokines that stimulate STAT-6 phosphorylation duringparenteral nutrition significantly increased levels of pIgR expressionand luminal IgA levels, indicating a cause and effect relationship. Inthis example, EEN decreased intestinal tissue levels of IL-4 andphosphorylated STAT-6, correlated with decreased pIgR and luminal sIgA.The addition of PAC to EEN at physiological levels (100 mg GAE/kg bodyweight) resulted in increased tissue IL-4, STAT-6 phosphorylation, pIgR,and luminal sIgA, supporting that PAC can influence health byinteracting with GALT function.

This is the first study to demonstrate that PAC supplementation canimprove luminal sIgA during EEN. PAC posed a significant challenge foraccurately quantifying luminal sIgA, because PAC form complexation withendogenous and dietary proteins, including immunoglobulins, throughhydrophobic and hydrogen bonding interaction. During analysis it wasobserved that the addition of small concentrations of PAC to luminalwash fluid from control animals rapidly decreased the detectable levelsof sIgA via ELISA quantification (unpublished observation). For thisreason, measurement of luminal sIgA in this study was achieved by firstdenaturing and reducing intestinal wash fluid samples with heat, sodiumdodecyl sulfate, and β-mercaptoethanol and performing western blotanalysis to detect the sIgA heavy chain directly. Future work with PACshould take the complexation and masking effect into consideration wheninvestigating intestinal sIgA.

In summary, this example shows that decreased enteral stimulation, suchas EEN or parenteral feeding, suppresses GALT function—including totalPP and lamina propria lymphocytes numbers, Th2 cytokine levels, and themucosal sIgA transport protein, pIgR—that leads to reduced luminal sIgAlevels. Consistent with the concept that PAC can provideimmunoprotective effects through interactions with the GALT andintestinal mucosa, the supplementation of physiological doses of PAC toEEN elevated GALT function and luminal sIgA compared to EEN feedingalone. This example indicates that moderate levels of PAC are beneficialwhen added to enteral diets by promotion of adaptive immune function.

Example 3 Enteral Proanthocyanidin Formulation

A liquid enteral formulation can be prepared to includeproanthocyanidins (1 μg/kg to 100 mg/kg; or about 8-100 mg/kg of patientbody weight; higher order polymers of PAC can be effective at lowerdosage ranges) and protein at about 25% of total calories by includingabout 87% of the protein from partially hydrolyzed casein and about 13%from one or more of the free amino acids arginine, cysteine, glutamine,ornithine, and proline. Carbohydrates can be included at about 35-40% ofcalories. Lipids can be added to comprise about 38-42% of calories, forexample, as a blend of medium chain triglycerides (50%), fish oil (25%),soy oil and/or soy lecithin (25%). Vitamin and mineral content can beformulated to meet daily requirements for a diet designed to provideapproximately 1500 calories (e.g., with approximately 1000 mL of theformulation). The proportions of the formulation can be adjusted higherfor increased caloric needs.

Example 4 Examples of Parenteral Nutrition Formulations

The tannins described herein can be added to enteral nutritionformulations such as that summarized in Table 4-2. The formulation canbe varied by adding triglycerides, e.g., including omega 3 and omega-6fatty acids, formulated to be administered in a parenteral nutritionformulation. Vitamins and minerals included in the multivitamin infusioncan include the ingredients described by Li et. al in “Effects ofparenteral and enteral nutrition on gut-associated lymphoid tissue”; J.Trauma 1995; 39:44-51 at Table 2. An exemplary parenteral formulation isshown in Table 4-1.

TABLE 4-1 Formulation of a parenteral nutrition solution (per 1 L).Component Amount Glucose 340 g Amino acids 44.7 g Sodium chloride 32 mEqSodium phosphate 36 mmol Potassium chloride 16 mEq Calcium gluconate37.5 mEq Potassium acetate 144 mEq Magnesium sulfate 8 mEq Manganese 0.8mg Copper 0.5 μg Zinc 2 mg Multivitamin infusion 10 mL

TABLE 4-2 Formulation of an elemental enteral nutrition solution (per 1L). Component Amount Glucose 356 g Amino acids (Clinisol) 60 g Sodiumchloride 32 mEq Sodium phosphate 36 mmol Potassium chloride 16 mEqCalcium gluconate 37.5 mEq Potassium acetate 44 mEq Magnesium sulfate 8mEq Manganese 0.8 mg Copper 0.5 μg Zinc 2.0 mg Vitamin C 200 mg VitaminA 3300 IU Vitamin D₃ 200 IU Thiamine 6 mg Riboflavan 3.6 mg PyridoxineHCl 6 mg Niacinamide 40 mg Folic Acid 600 mcg Biotin 60 mcgCyanocobalamin 5 mcg Vitamin E 10 IU (dl-α-tocopheryl Acetate) VitaminK₁ 150 mcg Dexpanthenol 15 mg

Example 5 Pharmaceutical Dosage Forms

The following formulations illustrate representative oral tannin dosageforms that may be used for the therapeutic or prophylacticadministration of a tannin described herein (hereinafter referred to as‘PAC’):

(i) Tablet 1 mg/tablet ‘PAC’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘PAC’ 20.0 Microcrystalline cellulose 410.0Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0

(iii) Capsule mg/capsule ‘PAC’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types oftannin. Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

Example 5 Cranberry Proanthocyanidins Preserves Microbiota DiversityDuring Elemental Enteral Nutrition

The human gastrointestinal tract is colonized by over 1000 bacterialspecies present in enormous quantities that are collectively termed themicrobiome (Guamer F. Role of intestinal flora in health and disease.Nutr Hosp. May 2007; 22 Suppl 2:14-19). The average microbiome maycontain 10 times the number of individual cells with over 100 times thegenetic diversity than its human host, highlighting the generallyunderappreciated role of microbes in human homeostasis and health. Themicrobiome plays fundamental roles in immune stimulation andmaintenance, digestion, and synthesis of vitamins and short chain fattyacids that are proposed to benefit the host and is therefore implicatedin influencing health (Guamer). Altered microbiome structures have beenidentified in states of metabolic dysfunction, such as obesity,diabetes, and inflammatory bowel diseases (Conterno L, Fava F, Viola R,Tuohy K M. Obesity and the gut microbiota: does up-regulating colonicfermentation protect against obesity and metabolic disease? Genes Nutr.August 2011; 6(3):241-260; and Maccaferri S, Biagi E, Brigidi P.Metagenomics: key to human gut microbiota. Dig Dis. 2011;29(6):525-530). One key modulatory force over microbiome structure isdietary intake, such as the ratio of carbohydrates to protein intake,dietary fiber, and polyphenolics. Under parenteral or enteral feedingwhere enteral bulk and complexity is decreased, especially in theproximal bowel, reduced microbiome diversity is measured (Kajiura T,Takeda T, Sakata S, et al. Change of intestinal microbiota withelemental diet and its impact on therapeutic effects in a murine modelof chronic colitis. Dig Dis Sci. September 2009; 54(9):1892-1900). Theloss of microbiome diversity is associated with reduced immuneparameters in the gastrointestinal tract, including decreased barrierfunction (Ott S J, Musfeldt M, Wenderoth D F, et al. Reduction indiversity of the colonic mucosa associated bacterial microflora inpatients with active inflammatory bowel disease. Gut. May 2004;53(5):685-693).

In investigating the role of dietary intake upon microbiome structure,recent attention has been drawn to the role of plant products, includingfiber and polyphenolics (de Vrese M, Schrezenmeir J. Probiotics,prebiotics, and synbiotics. Adv Biochem Eng Biotechnol. 2008; 111:1-66).Certain fermentable fibers, such as inulin and fructooligosacharides,are known prebiotics that are proposed to modulate the intestinalmicrobiome composition in a way that is beneficial or protective to thehost (de Vrese et al.). With the use of metabolomic analysis, microbiomemetabolism of polyphenolic compounds is also beginning to be elucidated(Tuohy K M, Conterno L, Gasperotti M, Viola R. Up-regulating the HumanIntestinal Microbiome Using Whole Plant Foods, Polyphenols, and/orFiber. J Agric Food Chem. September 2012; 60(36):8776-8782). In contrastto some polyphenolics that are largely metabolized before exiting thecolon, proanthocyanidins (PAC) remain intact during transit through thesmall bowel and are metabolized to lesser degrees in the large intestine(Ottaviani J I, Kwik-Uribe C, Keen C L, Schroeter H. Intake of dietaryprocyanidins does not contribute to the pool of circulating flavanols inhumans. Am J Clin Nutr. April 2012; 95(4):851-858). The studies outlinedin the examples above show the effect of supplementing elemental enteralnutrition (EEN) formulations with PAC to determine their effects upongut health, including alterations to the epithelial barrier and GALT. Inthose studies, samples of intestinal content from the ileal-cecaljunction were collected for crude analysis of the microbiome communityvia automated ribosomal intergenic spacer analysis (ARISA) to determinethe effect of EEN and EEN supplemented with PAC upon bacterialdiversity. ARISA is useful since the spacer between 16S and 23S rRNAgenes of microorganisms are commonly varied in length and sequence butwell conserved between species (Kovacs A, Yacoby K, Gophna U. Asystematic assessment of automated ribosomal intergenic spacer analysis(ARISA) as a tool for estimating bacterial richness. Res Microbiol.April 2010; 161(3):192-197). ARISA has been shown to highly correlatewith 16S sequencing as a tool for bacterial richness estimation (Kovacset al.).

Materials and Methods.

PAC Preparation, Animals, and Experimental Design.

The PAC preparation and characterization, animals, and experimentaldesign used were the same as described in Example 1 under “PACPreparation,” “PAC characterization by HPLC,” “PAC characterization bymatrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOFMS),” “Animals,” and “Experimental Design.” Additionally, during tissueharvest, luminal contents were collected with 1 mL of HBSS from the last1 cm of small intestine and stored at −80° C. until analysis.

ARISA.

Total genomic DNA was isolated from intestinal contents using DNeasyBlood and Tissue Kit (Qiagen, Hilden, Germany) and the 16S-23S rRNAspacer was amplified as previously described by Fisher and Triplett(Fisher M M, Triplett E W. Automated approach for ribosomal intergenicspacer analysis of microbial diversity and its application to freshwaterbacterial communities. Appl Environ Microbiol. October 1999;65(10):4630-4636). Briefly, PCR reactions were performed in duplicate in20 μl reactions with Taq DNA polymerase (Invitrogen, Carlsbad, Calif.),3 uM of MgCl₂, 2 μl 10×PCR buffer, 0.1 mM dNTP mix, nuclease-free water,and 1 pmol of primers. The reaction sequence was as follows: 3 min at94° C.; 30 cycles of 1 min denaturation at 94° C., 1 min annealing at53° C., 1.25 min elongation at 72° C.; and 10 min final elongation at72° C.

Fragment Analysis and Diversity Estimation.

A peak scanner (Applied Biosystems, Foster City, Calif.) with capillaryelectrophoresis was used to analyze total product peaks and data wererepresented as a fragment from at least one bacterial phylotype. Peaksobserved between 300 and 1000 base pairs with a threshold of at least100 fluorescent units were selected to make ARISA profiles. Data wereexported to Microsoft Excel for further analysis. Since the sum of thepeaks is proportional to the total DNA concentration, peaks at eachfragment size were calculated as a relative amount of total DNA. Peaksthat did not reach 0.5% of total DNA were removed from analysis in orderto minimize false peaks that might result in overestimation of speciesrichness. The remaining peaks were analyzed to estimate speciesdiversity using the Shannon's diversity index and Simpsons index ofdiversity (1-D) in PAST (http://folk.uio.no/ohammer/past/), which bothmeasure the richness and evenness of species present in samples.Significant differences were accepted at P<0.05. Next, treatment groupswere compared with the Jaccard similarity coefficient, used tostatistically compared similarity among groups. Finally, the peaks wereanalyzed using principal coordinate analysis (PCoA) (PAST), which aidsto visualize changes in community structure across a primary andsecondary axis and 95% confidence ellipses were determined.

Results

There were 53 total peaks (detected phylotypes) observed in Chow, whileonly 22 peaks were observed in EEN (Table 5-1). There were 40 totalpeaks observed in EEN+PAC. After removing peaks that did not reachgreater than 0.5% of total signal, there were 32 peaks in Chow (FIG.11A), 15 in EEN (FIG. 11B), and 25 in EEN+PAC (FIG. 11C). Overall, thepeaks appeared more similar between Chow and EEN+PAC and either groupcompared to EEN alone. This was tested across the entire data setstatically using peaks greater than 0.5% of total signal and calculatingJaccard's similarity coefficient. This similarity analysis revealed thatthe peak profiles of Chow and EEN+PAC are 65% similar to one another,while both dietary groups are only about 50% similar to EEN alone (FIG.12). To compare diversity, we calculated the Shannon's diversity index(SH) and Simpsons index of diversity (SI) between each group. Comparedwith Chow (SH 3.8; SI 0.891), EEN significantly reduced diversity (SH2.56, P<0.01; SI 0.85, P<0.05). Compared with EEN, the addition of PACto EEN significantly increased diversity (SH 3.46, P<0.01; SI 0.91,P<0.05). When comparing Chow to EEN+PAC, diversity differences weredetected with SH (P<0.05) but not SI (P=0.19). Finally, principalcoordinate analysis revealed slight shifts in composition using Unifracdistance metrics, but striking differences were observed since 95%confidence intervals overlapped (FIG. 13).

TABLE 5-1 Comparison of total, >1%, and >0.5% peaks observed (estimatedphylotypes) in ileum content samples measured via ARISA. Numbers Numbersof Numbers of of novel Numbers of phylotypes phylotypes phylotypesphylotypes present greater present greater relative Group present than1% than 0.5% to Chow Chow 53 33 32 — EEN 22 16 15 2 EEN + PAC 40 30 25 8Discussion

The relationship between diet, microbiome composition, and health isreceiving more attention as a testable hypothesis following thedevelopment of methods able to measure the interplay between bacterialcomposition and host metabolism (Tuohy et al.; Moco et al.).Polyphenolics are a prime target for these investigations sinceepidemiologic studies have repeatedly identified greater polyphenolicintake with lower incidence of chronic diseases (Scalbert A, Manach C,Morand C, Remesy C, Jimenez L. Dietary polyphenols and the prevention ofdiseases. Crit Rev Food Sci Nutr. 2005; 45(4):287-306). In this study,we employed a crude method of microbial analysis, ARISA, to determinethe effects of EEN or EEN supplemented with PAC upon microbiomediversity. We used ARISA since this method is cost effective andcorrelates well with more detailed sequencing methods to determinespecies diversity across samples (Kovacs et al.). Consistent withprevious work using EEN, we observed a decrease in microbiome diversitycompared with Chow feeding (Kajiura et al.). Interestingly, the additionof PAC to EEN resulted in a partial maintenance of diversity comparedwith EEN alone. These findings suggest PAC may support microbiomestructure as dietary compounds. These data build upon Examples 1 and 2,demonstrating beneficial effects of PAC supplementation to EEN upon theintestinal epithelium and GALT function.

Example 6 A-Type Proanthocyanidins (A-PAC), B-Type Proanthocyanidins(B-PAC), and Oligomeric Hydrolysable Tannins (OHT) Attenuate the Effectsof Elemental Enteral Nutrition on Size, Density, and Function ofIntestinal Goblet Cells in Mice

As outlined in Examples 1 and 2 above, addition of proanthocyanidintannins to a clinically used elemental enteral nutrition (EEN) solutioncounteracts the impairment of the intestinal immune barrier function. Inthose examples, a class of tannins, called A-type proanthocyanidins(A-PAC) (from cranberry fruit), were used. The present example showsthat other major types of tannins, B-type proanthocyanidins (B-PAC) andoligomeric hydrolyzable tannins (OHT), produce comparable effects whenadded to an EEN solution.

Materials and Methods

Experimental Methodology.

Cranberry fruit, grape seeds and pomegranate were used as sources forisolation of A-PAC, B-PAC and OHT, respectively. Isolation (liquidchromatography) and characterization (MALDI-TOF mass spectrometry)techniques were used to first separate the three distinct tanninpreparations. The isolation was performed as described in Example 1under “PAC Preparation,” except that grape seeds and pomegranate huskwere used for preparation of B-PAC and OHT, respectively. Cranberrypresscake was used for preparation of A-PAC. The characterization wasperformed as described in Example 1 under “PAC characterization bymatrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOFMS).” The effects of the three EEN formulations on the intestinal immunebarrier function were tested in a mouse model, as described in Example 1under “Animals” and “Experimental Design,” except that the A-PAC, B-PACor OHT was administered at a dose of 100 mg tannins/kg bodyweight/day.Analysis of ileal cytokines was performed as described in Example 1under “Analysis of ileal IL-4 and IL-13.” Analysis of luminal MUC2 wasperformed as described in Example 1 under “Analysis of luminal MUC2.”Histomorphometic analysis was performed as described in Example 1 under“Analysis of GC density and size.” Statistical analysis was performed asdescribed in Example 1 under “Statistical analysis.” Microbiotadiversity was performed using methods as described in Example 5.

Results.

Preliminary data indicates that, similar to the A-PAC cranberry tanninsshown in Example 1, the addition of B-PAC and OHT to enteral formulationsignificantly protects against the morphological atrophy of thegastrointestinal tissue induced by the consumption of the enteralformulation alone.

Discussion.

It is predicted that B-PAC tannins and OHT have each of the same effectsshown for A-PAC. Specifically, it is predicted that B-PAC tannins andOHT protect against EEN- and PN-dependent reductions in IL-4 and IL-13,goblet cell density and size, luminal MUC2 levels, Peyer's patchlymphocytes, phosphorylated STAT6, pIgR, sIgA, and microbiota diversity.It is surmised that a shared structural feature among the tannins(oligomeric phenolic structure), and not a distinct structural featureof the respective tannins (A-type interflavan bonds, B-type interflavanbonds, or ester bonds), is responsible for improving intestinal immunebarrier function. That is, the source of tannin is irrelevant.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method to attenuate suppressive immunologicaleffects of enteral nutrition on intestinal barrier function, the methodcomprising administering an effective amount of one or moreproanthocyanidins to a subject receiving the enteral nutrition, whereinthe enteral nutrition comprises tube feeding, wherein the subjectcomprises a mammal.
 2. A method to attenuate suppressive immunologicaleffects of enteral nutrition on intestinal barrier function, the methodcomprising administering an effective amount of one or moreproanthocyanidins to a subject receiving the enteral nutrition, whereinthe enteral nutrition comprises tube feeding, wherein the administeringcomprises administering a composition comprising the effective amount ofthe one or more proanthocyanidins and comprising no monomeric tannincomponents or less than about 5 wt. % monomeric tannin components.
 3. Amethod to attenuate suppressive immunological effects of enteralnutrition on intestinal barrier function, the method comprisingadministering an effective amount of one or more proanthocyanidins to asubject receiving the enteral nutrition, wherein the enteral nutritioncomprises tube feeding, wherein the one or more proanthocyanidins have adegree of polymerization of at least
 2. 4. A method to attenuatesuppressive immunological effects of enteral nutrition on intestinalbarrier function, the method comprising administering an effectiveamount of one or more proanthocyanidins to a subject receiving theenteral nutrition, wherein the enteral nutrition comprises tube feeding,wherein the method comprises administering the one or moreproanthocyanidins in an amount of from about 1 μg/kg of subject bodyweight to about 100 mg/kg of subject body weight, per day.
 5. The methodof claim 1 comprising administering the one or more proanthocyanidins inan amount of from about 5 mg/kg of subject body weight to about 10 mg/kgof subject body weight, per day.
 6. A method to attenuate suppressiveimmunological effects of enteral nutrition on intestinal barrierfunction, the method comprising administering an effective amount of oneor more proanthocyanidins to a subject receiving the enteral nutrition,wherein the enteral nutrition comprises tube feeding, wherein theeffective amount is an amount effective to result in one or more ofincreased levels of ileal IL-4, increased levels of ileal IL-13,increased density of goblet cells, increased goblet cell size, increasedrelative luminal MUC2 concentration, inhibited reduction ofphosphorylated STAT6, inhibited reduction of polymeric immunoglobulinreceptor (pIgR), and increased luminal secretory immunoglobulin-A (sIgA)levels compared to administering enteral nutrition without the effectiveamount of the one or more proanthocyanidins.